2&#39;-methoxy substituted oligomeric compounds and compositions for use in gene modulations

ABSTRACT

Compositions comprising first and second oligomers are provided wherein at least a portion of the first oligomer is capable of hybridizing with at least a portion of the second oligomer, at least a portion of the first oligomer is complementary to and capable of hybridizing to a selected target nucleic acid, and at least one of the first or second oligomers includes a modified sugar and/or backbone modification. In some embodiments the modification is a 2′-OCH 3  substituent group on a sugar moiety. Oligomer/protein compositions are also provided comprising an oligomer complementary to and capable of hybridizing to a selected target nucleic acid and at least one protein comprising at least a portion of an RNA-induced silencing complex (RISC), wherein at least one nucleotide of the oligomer has a modified sugar and/or backbone modification.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledISIS5314C4SEQ_ST25.txt created Dec. 21, 2017, which is 8 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides modified oligomers that modulate geneexpression via a RNA interference pathway. The oligomers of theinvention include one or more modifications thereon resulting indifferences in various physical properties and attributes compared towild type nucleic acids. The modified oligomers are used alone or incompositions to modulate the targeted nucleic acids. In preferredembodiments of the invention, the modifications include a 2′ substituentgroup on at least one sugar moiety of the oligomer.

BACKGROUND OF THE INVENTION

In many species, introduction of double-stranded RNA (dsRNA) inducespotent and specific gene silencing. This phenomenon occurs in bothplants and animals and has roles in viral defense and transposonsilencing mechanisms. This phenomenon was originally described more thana decade ago by researchers working with the petunia flower. Whiletrying to deepen the purple color of these flowers, Jorgensen et al.introduced a pigment-producing gene under the control of a powerfulpromoter. Instead of the expected deep purple color, many of the flowersappeared variegated or even white. Jorgensen named the observedphenomenon “cosuppression”, since the expression of both the introducedgene and the homologous endogenous gene was suppressed (Napoli et al.,Plant Cell, 1990, 2, 279-289; Jorgensen et al., Plant Mol. Biol., 1996,31, 957-973).

Cosuppression has since been found to occur in many species of plants,fungi, and has been particularly well characterized in Neurosporacrassa, where it is known as “quelling” (Cogoni and Macino, Genes Dev.2000, 10, 638-643; Guru, Nature, 2000, 404, 804-808).

The first evidence that dsRNA could lead to gene silencing in animalscame from work in the nematode, Caenorhabditis elegans. In 1995,researchers Guo and Kemphues were attempting to use antisense RNA toshut down expression of the par-1 gene in order to assess its function.As expected, injection of the antisense RNA disrupted expression ofpar-1, but quizzically, injection of the sense-strand control alsodisrupted expression (Guo and Kempheus, Cell, 1995, 81, 611-620). Thisresult was a puzzle until Fire et al. injected dsRNA (a mixture of bothsense and antisense strands) into C. elegans. This injection resulted inmuch more efficient silencing than injection of either the sense or theantisense strands alone. Injection of just a few molecules of dsRNA percell was sufficient to completely silence the homologous gene'sexpression. Furthermore, injection of dsRNA into the gut of the wormcaused gene silencing not only throughout the worm, but also in firstgeneration offspring (Fire et al., Nature, 1998, 391, 806-811).

The potency of this phenomenon led Timmons and Fire to explore thelimits of the dsRNA effects by feeding nematodes bacteria that had beenengineered to express dsRNA homologous to the C. elegans unc-22 gene.Surprisingly, these worms developed an unc-22 null-like phenotype(Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001,263, 103-112). Further work showed that soaking worms in dsRNA was alsoable to induce silencing (Tabara et al., Science, 1998, 282, 430-431).PCT publication WO 01/48183 discloses methods of inhibiting expressionof a target gene in a nematode worm involving feeding to the worm a foodorganism which is capable of producing a double-stranded RNA structurehaving a nucleotide sequence substantially identical to a portion of thetarget gene following ingestion of the food organism by the nematode, orby introducing a DNA capable of producing the double-stranded RNAstructure (Bogaert et al., 2001).

The posttranscriptional gene silencing defined in Caenorhabditis elegansresulting from exposure to double-stranded RNA (dsRNA) has since beendesignated as RNA interference (RNAi). This term has come to generalizeall forms of gene silencing involving dsRNA leading to thesequence-specific reduction of endogenous targeted mRNA levels; unlikeco-suppression, in which transgenic DNA leads to silencing of both thetransgene and the endogenous gene.

Introduction of exogenous double-stranded RNA (dsRNA) intoCaenorhabditis elegans has been shown to specifically and potentlydisrupt the activity of genes containing homologous sequences.Montgomery et al. suggests that the primary interference affects ofdsRNA are post-transcriptional. This conclusion being derived fromexamination of the primary DNA sequence after dsRNA-mediatedinterference and a finding of no evidence of alterations, followed bystudies involving alteration of an upstream operon having no effect onthe activity of its downstream gene.

These results argue against an effect on initiation or elongation oftranscription. Finally using in situ hybridization they observed thatdsRNA-mediated interference produced a substantial, although notcomplete, reduction in accumulation of nascent transcripts in thenucleus, while cytoplasmic accumulation of transcripts was virtuallyeliminated. These results indicate that the endogenous mRNA is theprimary target for interference and suggest a mechanism that degradesthe targeted mRNA before translation can occur. It was also found thatthis mechanism is not dependent on the SMG system, an mRNA surveillancesystem in C. elegans responsible for targeting and destroying aberrantmessages. The authors further suggest a model of how dsRNA mightfunction as a catalytic mechanism to target homologous mRNAs fordegradation. (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95,15502-15507).

Recently, the development of a cell-free system from syncytialblastoderm Drosophila embryos, which recapitulates many of the featuresof RNAi, has been reported. The interference observed in this reactionis sequence specific, is promoted by dsRNA but not single-stranded RNA,functions by specific mRNA degradation, and requires a minimum length ofdsRNA. Furthermore, preincubation of dsRNA potentiates its activitydemonstrating that RNAi can be mediated by sequence-specific processesin soluble reactions (Tuschl et al., Genes Dev., 1999, 13, 3191-3197).

In subsequent experiments, Tuschl et al, using the Drosophila in vitrosystem, demonstrated that 21- and 22-nt RNA fragments are thesequence-specific mediators of RNAi. These fragments, which they termedshort interfering RNAs (siRNAs), were shown to be generated by an RNaseIII-like processing reaction from long dsRNA. They also showed thatchemically synthesized siRNA duplexes with overhanging 3′ ends mediateefficient target RNA cleavage in the Drosophila lysate, and that thecleavage site is located near the center of the region spanned by theguiding siRNA. In addition, they suggest that the direction of dsRNAprocessing determines whether sense or antisense target RNA can becleaved by the siRNA-protein complex (Elbashir et al., Genes Dev., 2001,15, 188-200). Further characterization of the suppression of expressionof endogenous and heterologous genes caused by the 21-23 nucleotidesiRNAs have been investigated in several mammalian cell lines, includinghuman embryonic kidney (293) and HeLa cells (Elbashir et al., Nature,2001, 411, 494-498).

The Drosophila embryo extract system has been exploited, using greenfluorescent protein and luciferase tagged siRNAs, to demonstrate thatsiRNAs can serve as primers to transform the target mRNA into dsRNA. Thenascent dsRNA is degraded to eliminate the incorporated target mRNAwhile generating new siRNAs in a cycle of dsRNA synthesis anddegradation. Evidence is also presented that mRNA-dependent siRNAincorporation to form dsRNA is carried out by an RNA-dependent RNApolymerase activity (RdRP) (Lipardi et al., Cell, 2001, 107, 297-307).

The involvement of an RNA-directed RNA polymerase and siRNA primers asreported by Lipardi et al. (Lipardi et al., Cell, 2001, 107, 297-307) isone of the many intriguing features of gene silencing by RNAinterference. This suggests an apparent catalytic nature to thephenomenon. New biochemical and genetic evidence reported by Nishikuraet al. also shows that an RNA-directed RNA polymerase chain reaction,primed by siRNA, amplifies the interference caused by a small amount of“trigger” dsRNA (Nishikura, Cell, 2001, 107, 415-418).

Investigating the role of “trigger” RNA amplification during RNAinterference (RNAi) in Caenorhabditis elegans, Sijen et al revealed asubstantial fraction of siRNAs that cannot derive directly from inputdsRNA. Instead, a population of siRNAs (termed secondary siRNAs)appeared to derive from the action of the previously reported cellularRNA-directed RNA polymerase (RdRP) on mRNAs that are being targeted bythe RNAi mechanism. The distribution of secondary siRNAs exhibited adistinct polarity (5′-3′; on the antisense strand), suggesting a cyclicamplification process in which RdRP is primed by existing siRNAs. Thisamplification mechanism substantially augmented the potency ofRNAi-based surveillance, while ensuring that the RNAi machinery willfocus on expressed mRNAs (Sijen et al., Cell, 2001, 107, 465-476).

Most recently, Tijsterman et al. have shown that, in fact,single-stranded RNA oligomers of antisense polarity can be potentinducers of gene silencing. As is the case for co-suppression, theyshowed that antisense RNAs act independently of the RNAi genes rde-1 andrde-4 but require the mutator/RNAi gene mut-7 and a putative DEAD boxRNA helicase, mut-14. According to the authors, their data favor thehypothesis that gene silencing is accomplished by RNA primer extensionusing the mRNA as template, leading to dsRNA that is subsequentlydegraded suggesting that single-stranded RNA oligomers are ultimatelyresponsible for the RNAi phenomenon (Tijsterman et al., Science, 2002,295, 694-697).

Several recent publications have described the structural requirementsfor the dsRNA trigger required for RNAi activity. Recent reports haveindicated that ideal dsRNA sequences are 21 nt in length containing 2 nt3′-end overhangs (Elbashir et al, EMBO (2001), 20, 6877-6887, SabineBrantl, Biochimica et Biophysica Acta, 2002, 1575, 15-25.) In thissystem, substitution of the 4 nucleosides from the 3′-end with2′-deoxynucleosides has been demonstrated to not affect activity. On theother hand, substitution with 2′-deoxynucleosides or 2′-OCH₃-nucleosidesthroughout the sequence (sense or antisense) was shown to be deleteriousto RNAi activity.

Investigation of the structural requirements for RNA silencing in C.elegans has demonstrated modification of the internucleotide linkage(phosphorothioate) to not interfere with activity (Parrish et al.,Molecular Cell, 2000, 6, 1077-1087.) It was also shown by Parrish etal., that chemical modification like 2′-amino or 5-iodouridine are welltolerated in the sense strand but not the antisense strand of the dsRNAsuggesting differing roles for the 2 strands in RNAi. Base modificationsuch as guanine to inosine (where one hydrogen bond is lost) has beendemonstrated to decrease RNAi activity independently of the position ofthe modification (sense or antisense). Some “position independent” lossof activity has been observed following the introduction of mismatchesin the dsRNA trigger. Some types of modifications, for exampleintroduction of sterically demanding bases such as 5-iodoU, have beenshown to be deleterious to RNAi activity when positioned in theantisense strand, whereas modifications positioned in the sense strandwere shown to be less detrimental to RNAi activity. As was the case forthe 21 nt dsRNA sequences, RNA-DNA heteroduplexes did not serve astriggers for RNAi. However, dsRNA containing 2′-F-2′-deoxynucleosidesappeared to be efficient in triggering RNAi response independent of theposition (sense or antisense) of the 2′-F-2′-deoxynucleosides.

In one study the reduction of gene expression was studied usingelectroporated dsRNA and a 25mer morpholino oligomer in postimplantation mouse embryos (Mellitzer et al., Mechanisms of Development,2002, 118, 57-63). The morpholino oligomer did show activity but was notas effective as the dsRNA.

A number of PCT applications have recently been published that relate tothe RNAi phenomenon. These include: PCT publication WO 00/44895; PCTpublication WO 00/49035; PCT publication WO 00/63364; PCT publication WO01/36641; PCT publication WO 01/36646; PCT publication WO 99/32619; PCTpublication WO 00/44914; PCT publication WO 01/29058; and PCTpublication WO 01/75164.

U.S. Pat. Nos. 5,898,031 and 6,107,094, each of which is commonly ownedwith this application and each of which is herein incorporated byreference, describe certain oligonucleotide having RNA like properties.When hybridized with RNA, these oligonucleotides serve as substrates fora dsRNase enzyme with resultant cleavage of the RNA by the enzyme.

In another recently published paper (Martinez et al., Cell, 2002, 110,563-574) it was shown that single stranded as well as double strandedsiRNA resides in the RNA-induced silencing complex (RISC) together withelF2C1 and elf2C2 (human GERp950) Argonaute proteins. The activity of5′-phosphorylated single stranded siRNA was comparable to the doublestranded siRNA in the system studied. In a related study, the inclusionof a 5′-phosphate moiety was shown to enhance activity of siRNA's invivo in Drosophilia embryos (Boutla, et al., Curr. Biol., 2001, 11,1776-1780). In another study, it was reported that the 5′-phosphate wasrequired for siRNA function in human HeLa cells (Schwarz et al.,Molecular Cell, 2002, 10, 537-548).

In yet another recently published paper (Chiu et al., Molecular Cell,2002, 10, 549-561) it was shown that the 5′-hydroxyl group of the siRNAis essential as it is phosphorylated for activity while the 3′-hydroxylgroup is not essential and tolerates substitute groups such as biotin.It was further shown that bulge structures in one or both of the senseor antisense strands either abolished or severely lowered the activityrelative to the unmodified siRNA duplex. Also shown was severe loweringof activity when psoralen was used to cross link an siRNA duplex.

Like the RNAse H pathway, the RNA interference pathway for modulation ofgene expression is an effective means for modulating the levels ofspecific gene products and, thus, would be useful in a number oftherapeutic, diagnostic, and research applications involving genesilencing.

The present invention therefore provides oligomeric compounds useful formodulating gene expression pathways, including those relying onmechanisms of action such as RNA interference and dsRNA enzymes, as wellas antisense and non-antisense mechanisms. One having skill in the art,once armed with this disclosure will be able, without undueexperimentation, to identify preferred oligonucleotide compounds forthese uses.

SUMMARY OF THE INVENTION

In certain aspects, the invention relates to compositions comprising afirst oligomer and a second oligomer, each having linked nucleosidicbases. At least a portion of the first oligomer is capable ofhybridizing with at least a portion of the second oligomer, at least aportion of the first oligomer is complementary to and capable ofhybridizing to a selected target nucleic acid, and at least one of thefirst and second oligomers includes at least one sugar moiety having a2′-OCH₃ substituent group.

In some embodiments, the first and second oligomers are a complementarypair of siRNA oligomers. In certain embodiments, the first and secondoligomers are an antisense/sense pair of oligomers. In somecompositions, each of the first and second oligomers has about 0 toabout 40 linked nucleosides. In other compositions, each of the firstand second oligomers has about 18 to about 30 linked nucleosides. In yetother compositions, each of the first and second oligomers has about 21to about 24 linked nucleosides.

In certain aspects, the first oligomer is an antisense oligomer. Inother aspects, the second oligomer comprises a sense oligomer. In someembodiments, the second oligomer has a plurality of ribose nucleosideunits. In still other embodiments, the first oligomer includes said2′-OCH₃ substituent group.

In some preferred embodiments, the first oligomer comprises a 3′terminus and a 5′ terminus, said 5′ terminus comprising a hydroxyterminal group. In other preferred embodiments, the 2′-OCH₃ substitutentgroup extend from the 3′ terminus of the first oligomer through thefourth, third, or penultimate nucleoside from the 5′ terminus.

In certain preferred embodiments, the first oligomer comprises a 3′terminus and a 5′ terminus, wherein the nucleoside at the 5′ terminuscomprises a 2′-OCH₃ substitutent group. In some of these compositions,the 5′ terminus comprises a 3′-phosphate terminal group.

In some aspects, the first oligomer is a chimeric oligomeric compound.Certain chimeric compositions are a gapmer, an inverted gapmer, a3′-hemimer, a 5′-hemimer or a blockmer. In some aspects, the chimericoligomeric compound comprises two terminal RNA segments havingnucleosides of a first type and an internal RNA segment havingnucleosides of a second type and where said nucleosides of said firsttype are different from said nucleosides of said second type. In certainpreferred embodiments, the nucleosides of said first type includes a2′-OCH₃ substitutent group.

In other embodiments, the invention is directed tooligonucleomer/protein compositions comprising an oligomer complementaryto and capable of hybridizing to a selected target nucleic acid, and atleast one protein comprising at least a portion of a RNA-inducedsilencing complex (RISC). The oligomer includes at least one nucleosidehaving a 2′-OCH₃ substituent group on the sugar moiety.

Also provided by the present invention are pharmaceutical compositionscomprising any of the above compositions or oligomeric compounds and apharmaceutically acceptable carrier.

Methods for modulating the expression of a target nucleic acid in a cellare also provided, wherein the methods comprise contacting the cell withany of the above compositions or oligomeric compounds.

Methods of treating or preventing a disease or condition associated witha target nucleic acid are also provided, wherein the methods compriseadministering to a patient having or predisposed to the disease orcondition a therapeutically effective amount of any of the abovecompositions or oligomeric compounds.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides oligomeric compounds useful in themodulation of gene expression. Although not intending to be bound bytheory, oligomeric compounds of the invention modulate gene expressionby hybridizing to a nucleic acid target resulting in loss of normalfunction of the target nucleic acid. As used herein, the term “targetnucleic acid” or “nucleic acid target” is used for convenience toencompass any nucleic acid capable of being targeted including withoutlimitation DNA, RNA (including pre-mRNA and mRNA or portions thereof)transcribed from such DNA, and also cDNA derived from such RNA. In apreferred embodiment of this invention modulation of gene expression iseffected via modulation of a RNA associated with the particular geneRNA.

The invention provides for modulation of a target nucleic acid that is amessenger RNA. The messenger RNA is degraded by the RNA interferencemechanism as well as other mechanisms in which double stranded RNA/RNAstructures are recognized and degraded, cleaved or otherwise renderedinoperable.

The functions of RNA to be interfered with can include replication andtranscription.

Replication and transcription, for example, can be from an endogenouscellular template, a vector, a plasmid construct or otherwise. Thefunctions of RNA to be interfered with can include functions such astranslocation of the RNA to a site of protein translation, translocationof the RNA to sites within the cell which are distant from the site ofRNA synthesis, translation of protein from the RNA, splicing of the RNAto yield one or more RNA species, and catalytic activity or complexformation involving the RNA which may be engaged in or facilitated bythe RNA. In the context of the present invention, “modulation” and“modulation of expression” mean either an increase (stimulation) or adecrease (inhibition) in the amount or levels of a nucleic acid moleculeencoding the gene, e.g., DNA or RNA. Inhibition is often the preferredform of modulation of expression and mRNA is often a preferred targetnucleic acid.

Compounds of the Invention

The compounds of the invention include oligomeric compounds thatcomprise at least one monomeric unit that has a 2′-OCH₃ substituentgroup on the sugar moiety. 2′-Sugar substituents are described in moredetail in U.S. Pat. Nos. 5,670,633, 5,914,396, 6,005,087, 6,222,025,6,307,040, 6,531,584 and in U.S. patent application Ser. No. 10/444,628.The disclosure of each of these patents and applications is incorporatedherein by reference in its entirety.

In some compositions, one or more sugars may contain a 2′-substitutentother than 2′-OCH₃. In certain embodiments, these substituent group maybe halogen, amino, trifluoroalkyl, trifluoroalkoxy, azido, aminooxy,alkyl, alkenyl, alkynyl, O-, S-, or N(R*)-alkyl; O-, S-, orN(R*)-alkenyl; O-, S- or N(R*)-alkynyl; O-, S- or N-aryl, O-, S-, orN(R*)-aralkyl; wherein said alkyl, alkenyl, alkynyl, aryl and aralkylmay be substituted or unsubstituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl,C₂ to C₁₀ alkynyl, C₅-C₂₀ aryl or C₆-C₂₀ aralkyl; and said substitutedC₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₂ to C₁₀ alkynyl, C₅-C₂₀ aryl orC₆-C₂₀ aralkyl comprising substitution with hydroxy, alkoxy, thioalkoxy,phthalimido, halogen, amino, keto, carboxyl, nitro, nitroso, cyano,aryl, haloalkyl, haloalkoxy, imidazole, azido, hydrazino, aminooxy,isocyanato, sulfoxide, sulfone, disulfide, silyl, heterocycle,carbocycle, an intercalator, a reporter group, a conjugate, a polyamine,a polyamide, a polyalkylene glycol, or a polyether of the formula(—O-alkyl)_(m), where m is 1 to about 10; and R* is hydrogen, or aprotecting group.

As discussed above, the 2′ substituent may be a halogen. In somepreferred embodiments the halogen is F. Certain oligonucleosides thatare N3′->P5′ phosphoramidates having 2′ fluoro substituents have beenshown to have superior acid stability. These compositions can be made byprocedures taught is U.S. Pat. No. 5,684,143, the disclosure of which isincorporated herein in its entirety.

Some preferred substituents are 2′-O-alkyl substituents. These alkylgroups include lower alkyl groups having from about 1 to about 6 carbonatoms. In some preferred embodiments, the alkyl is a C₂-C₆ group. Other2′ substituent groups include 2′-methoxyethoxy (MOE, 2′-OCH₂CH₂OCH₃) and2′-O-[2-(2-N,N-dimethylaminoethyl)oxyethyl]. These substituents aredescribed in U.S. Pat. Nos. 6,043,352 and 6,005,094, the disclosures ofwhich are incorporated herein by reference in their entirety.

In other embodiments, the 2′ substituent is —O—R²⁶-thio-R²⁶ or—C—R²⁶-thio-R²⁶, wherein said R²⁶ is independently a compound selectedfrom a group consisting of alkyl, allyl, alkenyl, alkynyl, aryl,alkylaryl, carbocyclic aryl, heterocyclic aryl, amide and ester. In someembodiments, R²⁶ is alkyl, allyl, alkenyl, alkynyl, aryl, alkylaryl,carbocyclic aryl, or heterocyclic aryl. In certain embodiments, the 2′substituent is 2′-O-methylthiomethyl. In other embodiments, the 2′substituent is 2′-O-methylthioethyl. These substituents are described inU.S. Pat. Nos. 5,716,824, 5,840,876 and 6,239,272, the disclosures ofwhich are incorporated by reference herein in their entirety.

In certain embodiments, the 2′ substituent is cyano, fluoromethyl,thioalkoxyl, fluoroalkoxyl, alkylsulfinyl, alkylsulfonyl, allyloxy oralkeneoxy. In other embodiments, the 2′-substituent is 2′-alkylsulfinylor alkylsulfonyl. In other preferred embodiments, the 2′-substituent is2′-thioalkoxyl, preferably, a 2′-S—(C₁-C₂₀ alkyl) substituent. Thesesubstituents are described in U.S. Pat. No. 5,859,221, the disclosure ofwhich is incorporated herein by reference.

Some 2′ substituents useful in the invention are of the formula —X—Y. Xis O, S, NR²⁷, or CR²⁷ ₂ wherein each R is independently H or C₁₋₆alkyl. Y is a linker moiety, a drug residue optionally attached througha linker moiety, a label optionally attached through a linker moiety, ora property-affecting group optionally attached through a linker moiety.In some embodiments, Y is a drug moiety. In certain embodiments, thedrug moiety is selected from the group consisting of netropsin,anthramycin, quinoxaline antibiotics, actinomycin, and pyrrolo (1-4)benzodiazepine. In other embodiments, Y is substituted or unsubstitutedalkyl (C₂₋₂₀), substituted or unsubstituted alkenyl (C₂₋₂₀), substitutedor unsubstituted aryl (C₆₋₂₀), wherein the substituents are selectedfrom the group consisting of a hydroxyl, an amino, a mercaptyl, acarboxy or a keto moiety, —CH₂COOH, —CH₂COONH₂, —CH₂COOEt,—CH₂CONHCH₂CH₂NH₂ and SiR²⁸ ₃ wherein R²⁸ is alkyl (C₂₋₆). In certainpreferred embodiments, X is O or S. These substituents are described inU.S. Pat. Nos. 5,466,786 and 5,792,847, the disclosures of which areincorporated herein by reference in their entirety.

Certain 2′ substituents are of the formula:

-G1-G2-G3 where G1 is a bivalent linker, G2 is an aryl or heteroaryl oraryl or heteroaryl containing group and G3 is an RNA cleaving moietyhaving, for example, general acid/base properties. In certain furtherpreferred embodiments of the inventions, G3 further includes anelectrophilic catalyst.

The bivalent liker may be a mono- or polyatomic linker. In someembodiments, the bivalent linker is of the formula -G¹¹-G¹². G¹¹contains a heteroatom and G¹² contains an alkyl, alkenyl or alkynylgroup. Preferred heteroatoms include O, S, and N—H or N-alkyl. In someembodiments, the linker may be methylene groups—i.e., —(CH₂)_(n)— or mayinclude heteroatoms and functional groups, e.g., —CH₂OCH₂CH₂O— or—CH₂O—CH₂CH₂NH— or —COOCH₂CH₂O—. In certain embodiments of theinvention, G1 connects to the internucleoside linkage, i.e. the sugarlinking group.

G2 preferably is a polycyclic moiety having from 2 to 6 rings, at least2 of said rings being joined to form an electronically conjugatedsystem. Representative G2 groups include naphthalene, anthracene,phenanthrene, benzonaphthalene, fluorene, carbazole,pyrido[4,3-b]carbazole, acridine, pyrene, anthraquinone, quinoline,phenylquinoline, xanthene or 2,7-diazaanthracene groups. Structures ofthis type preferably act as intercalators. Other intercalators believedto be useful are described by Denny, Anti-Cancer Drug Design 1989, 4,241.

RNA-cleaving group G3 can be a functionality that has both general acidand general base characteristics. It also can possess electrophiliccatalytic characteristics. It can further possess metal ion coordinatingcharacteristics. Such substituents are described in U.S. Pat. No.6,358,931, whose disclosure is incorporated by reference herein in itsentirety.

The 2′ substituent may be of the formula —O-G1-G2-G3 where G1 is alkyl,alkenyl, or alkynyl; G2 is an aryl; and G3 includes at least oneimidazole. In some preferred embodiments, G3 is an imidazole or abis-imidazole moiety. In other embodiments, G1-G2-G3 is an alkynylmoiety. These substituents are described in U.S. Pat. No. 5,514,786,whose disclosure is incorporated herein by reference in its entirety.

Some useful 2′ substituents are of the formula —C(X)—N(R²⁹)(R³⁰)

where:R²⁹ and R³⁰, independently, are H, R³³, R³⁴, an amine protecting groupor have formula R³³—N(R³¹)(R³²), C(X)—R³³, C(X)—R³⁴—R³³, C(X)-Q-R³⁴—R³³,or C(X)-Q-R³³; R³¹ and R³², independently, are H, R³³, R³⁴, an amineprotecting group or have formula C(X)—R³³, C(X)—R³⁴—R³³, C(X)-Q-R³⁴—R³³,or C(X)-Q-R³³;R₃₃ is a steroid molecule, biotin, dinitrophenyl, a fluorescein dye, alipophilic molecule, a reporter enzyme, a peptide, a protein, includesfolic acid, or has formula -Q-(CH₂CH₂-Q-)_(x)-R³⁵;R³⁴ is alkyl having from 1 to about 10 carbon atoms;

X is O or S;

each Q is, independently, is NH, O, or S;R³⁵ is H, R³⁴, C(O)OH, C(O)OR³⁴, C(O)R⁴¹, R³⁴—N₃, or R³⁴—NH₂;R⁴¹ is Cl, Br, I, SO₂R⁴² or has structure:

m is 2 or 7; andR⁴² alkyl having 1 to about 10 carbon atoms.

In some embodiments, R³⁴ is alkyl having 1 to about 10 carbon atoms,preferably having 6 carbon atoms. In other embodiments, R²⁹ is H and R³⁰is R³³. Some compounds are such that R²⁹ is H and R³⁰ is alkyl having 1to about 10 carbon atoms, preferably 1 or 2 carbon atoms. In certainembodiments, R²⁹ and R³⁰, together, are phthalimido. In otherembodiments, R²⁹ is H and R³⁰ is R³⁴—N(R³¹)(R³²). These compounds aredescribed in U.S. Pat. No. 6,111,085, whose disclosure is incorporatedherein by reference in its entirety.

The sugar substituent may be a 2′-aminoalkoxy or a 2′-imidazolylalkoxysubstituent, wherein the alkoxy moiety of said substituent is C₁-C₂₀. Insome embodiments the substituent is 2′-O-(aminoprop-3-yl) or2′-O-(aminobut-4-yl). In other embodiments, the substituent is2′-O->(imidazol-1-yl) prop-3-yl or 2′-O->(imidazol-1-yl) but-4-yl. See,for example, U.S. Pat. No. 5,872,232, which is incorporated herein byreference in its entirety.

Certain 2′ modifications are groups of the formula:

where m is from 0 to 10; y is from 1 to 10; x is 1; E is N(R³⁷)(R³⁸) orN═C(R³⁷)(R³⁸); and each of R³⁶, R³⁷ and R³⁸ is, independently, H, C₁-C₁₀alkyl, and an amino protecting group, or R³⁷ and R³⁸ together, are anamino protecting group or wherein R³⁷ and R³⁸ are joined in a C₄-C₁₀ring structure that can include at least one heteroatom selected from Nand O. In certain embodiments, R³⁷ and R³⁸ are independently H or C₁-C₁₀alkyl. In other embodiments R³⁷ and R³⁸ are joined in a C₄-C₁₀ ringstructure that optionally includes one or more heteroatoms selected fromN and O. Some compounds of the invention comprise a ring structure thatis an imidazolyl ring, a piperidinyl ring, a morpholinyl ring or asubstituted piperazinyl ring. Certain piperazine may be optionallysubstituted with a C₁-C₁₂ alkyl. These substituent groups may beproduced by methods taught by U.S. Pat. Nos. 6,127,533 and 6,172,209 thedisclosures of which are incorporated by reference herein in theirentirety.

In certain embodiments, the 2′ substituent is of the formula:

where R³⁹ is alkyl having from 1 to about 10 carbon atoms or(CH₂—CH₂-Q)_(x);R⁴⁰ is alkenyl having 2 to about 10 carbon atoms;R²⁹ and R³⁰, independently, are H, R³³, R³⁹, an amine protecting groupor have formula R³⁹—N(R³¹)(R³²), C(X)—R³³, C(X)—R³⁹—R³³, C(X)-Q-R³⁹—R³³,or C(X)-Q-R³³;R³¹ and R³², independently, are H, R³³, R³⁹, an amine protecting groupor have formula C(X)—R³³, C(X)—R³⁹—R³³, C(X)-Q-R³⁹—R³³, or C(X)-Q-R³³;R³³ is a steroid molecule, a reporter molecule, a lipophilic molecule, areporter enzyme, a peptide, a protein, includes folic acid, or hasformula -Q-(CH₂CH₂-Q-)_(x)-R³⁵;

X is O or S;

each Q is, independently, is NH, O, or S;x is 1 to about 200;R³⁵ is H, R³⁹, C(O)OH, C(O)OR³⁹, C(O)R⁴¹, R³⁹—N₃, or R³⁹—NH₂;R⁴¹ is Cl, Br, I, SO₂R⁴² or has structure:

m is 2 or 7; and R₄₂ is an alkyl having 1 to about 10 carbon atoms.In some embodiments, R³⁹ is alkyl having 6 carbon atoms. In certainembodiments, R⁴⁰ is 2-propenyl. In other embodiments, R²⁹ is H and R³⁰is H. In yet other embodiments, R²⁹ is H and R³⁰ is R³³. In somecompositions, R²⁹ is H and R³⁰ is alkyl having 1 to about 10 carbonatoms. In certain embodiments, R²⁹ and R³⁰, together, are phthalimido.In other embodiments, R²⁹ is H and R³⁰ is R³⁹—N(R³¹)(R³²). In certaincompositions of the invention, R³¹ is H and R³² is R³³. In othercompositions, R³¹ is H and R³² is alkyl having 1 to about 10 carbonatoms. In yet other compositions, R³⁰ is H and R³² is an alkyl having 1or 2 carbon atoms. In yet other compositions, R³¹ and R³², together, arephthalimido. These substituents may be synthesized by methods taught byU.S. Pat. No. 6,166,188, the disclosure of which is incorporated hereinby reference in its entirety.

Some 2′ substituents are of the formula:

wherein each E₁ and E₂ is, independently, H, C₁-C₁₀ alkyl, C₂-C₁₀alkenyl, C₂-C₁₀ alkynyl, C₆-C₁₄ aryl, (CH₂)_(m)—S—R⁴³ where m is from 1to 10, —{(CH₂)_(nn)—N(H)}_(nnn)—(CH₂)_(nn)NH₂ where each nn is aninteger from 2 to 4 and nnn is an integer from 2 to 10, a polypeptidehaving from 2 to 10 peptide linked amino acids, a folic acid moietyoptionally bearing a linking group attaching said folic acid moiety fromthe α or γ carboxyl group to the 2′-substituent wherein said linkinggroup is —N(H)—(CH₂)₆—, or a cholesterol moiety optionally bearing alinking group attaching said cholesterol moiety from the hydroxyl groupto the 2′-substituent, wherein said linking group is—C(═O)—N(H)—(CH₂)₆—; and R⁴³ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀alkynyl, C₆-C₁₄ aryl or a thio protecting group. In some embodiments,each E₁ and E₂ is independently C₁-C₁₀ alkyl.

In some embodiments, each E₁ and E₂ is, independently, C₁-C₁₀ alkyl, orone of E₁ and E₂ is H and the other of E₁ and E₂ is —CH₃;

or each E₁ and E₂ is, independently, H, —(CH₂)_(m)—S—R⁴³ where m is from1 to 10, —{(CH₂)_(nn)—N(H)}_(nnn)—(CH₂)_(n)NH₂ where each nn is from 2to 4 and nnn is from 2 to 10, a polypeptide having from 2 to 10 peptidelinked amino acids, a folic acid moiety optionally bearing a linkinggroup attaching said folic acid moiety from the α or γ carboxyl group tothe 2′-substituent wherein said linking group is —N(H)—(CH₂)₆—, or acholesterol moiety optionally bearing a linking group attaching saidcholesterol moiety from the hydroxyl group to the 2′-substituent,wherein said linking group is —C(═O)—N(H)—(CH₂)₆—, provided that onlyone of E₁ and E₂ is H; and R⁴³ is H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, C₆-C₁₄ aryl or a thio protecting group.

In certain embodiments, E₁ is H and E₂ is —(CH₂)_(m)—S—R⁴³. In otherembodiments, R⁴³ is C₁-C₁₀ alkyl. Further embodiments are those whereR⁴³ is methyl. In yet other embodiments, E₂ is {(CH₂)_(nn)—N(H)}nnn(CH₂)_(nn)NH₂ where each nn is from 2 to 4 and nnn is from 2 to 10. Insome compositions, E₂ is —(CH₂)₃—N(H)—(CH₂)₄—N(H)—(CH₂)₃—NH₂ or—(CH₂)₄—N(H)—(CH₂)₃—NH₂. In certain embodiments, E₂ is said polypeptide.In some embodiments, the polypeptide is Lys-Tyr-Lys, Lys-Trp-Lys orLys-Lys-Lys-Lys. In yet other embodiments, E₂ is a linked folic acid or5-methyl-tetrahydrofolic acid moiety. In further embodiments, E₂ is acholesterol moiety. These substituents may be made may methods disclosedin U.S. Pat. No. 6,147,200, whose disclosure is incorporated byreference herein in its entirety.

Other 2′ substituent groups of the invention are of the formula:

—X—(CR⁷¹R⁷²)_(n)—O—SO₂—O⁻-Y⁺

where X is a O, S, or N; R⁷¹ and R⁷² are independently H, alkyl, aryl,O-alkyl, O-aryl, carboxylic acid, amide, ester, halogen,trifluoromethyl, or amine; n is an integer from about 2 to about 10;and, Y is H, Li, Na, K, Cs or an amine. The synthesis of compounds withthese substituent groups is described in U.S. Pat. No. 6,227,982, thedisclosure of which is incorporated herein in its entirety.

Some 2′ substituents are of the formula:

where:each Z is, independently, a single bond, O, N or S;each R⁴⁶, R⁴⁷, R⁴⁸, and R⁴⁹ is, independently, hydrogen, C(O)R⁵⁰,substituted or unsubstituted C₁-C₁₀ alkyl, substituted or unsubstitutedC₂-C₁₀ alkenyl, substituted or unsubstituted C₂-C₁₀ alkynyl,alkylsulfonyl, arylsulfonyl, a chemical functional group or a conjugategroup, wherein the substituent groups are selected from hydroxyl, amino,alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen,alkyl, aryl, alkenyl and alkynyl;or R⁴⁶ and R⁴⁷, together, are R⁵¹;each R⁵⁰ is, independently, substituted or unsubstituted C₁-C₁₀ alkyl,trifluoromethyl, cyanoethyloxy, methoxy, ethoxy, t-butoxy, allyloxy,9-fluorenylmethoxy, 2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy,benzyloxy, butyryl, iso-butyryl, phenyl or aryl;each R⁵¹ is, independently, hydrogen or forms a phthalimide moiety withthe nitrogen atom to which it is attached;each m is, independently, zero or 1; andeach n is, independently, an integer from 1 to about 6. Thesesubstituents may be synthesized by methods taught in U.S. Pat. No.6,534,639, whose disclosure is incorporated herein by reference in itsentirety.

In certain embodiments, the 2′ modification is of the formula —OR⁵²where R⁵² is

where R⁵³ is hydrogen, C₁-C₂₁ alkyl, C₂-C₂₁ alkenyl, C₂-C₂₁ alkynyl or—C(═O)-alkyl; R⁵⁴ is hydrogen, C₁-C₁₀ alkyl, —CH₂—O—R⁵⁵ or a radical offormula Ib; R⁵⁵ is hydrogen, C₁-C₂₂ alkyl, C₃-C₂₁ alkenyl, or partiallyor completely fluorine-substituted C₁-C₁₀ alkyl or —[(CH₂)₂—O]_(m)—R⁵⁶;R⁵⁶ is hydrogen or C₁-C₂₁ alkyl; Z is —(CH₂)_(p)— or—(CH₂—CH₂—O)_(q)—CH₂CH₂—, it being possible for Z in the case of —CH₂—to be unsubstituted or substituted by one or more identical or differentsubstituents selected from C₁-C₁₀ alkyl, C₅-C₆ cycloalkyl andunsubstituted or C₁-C₄ alkyl-substituted phenyl; n is an integer from 1to 12; m is an integer from 1 to 4; p is an integer from 1 to 10; and qis an integer from 1 to 4. Such substituents are described in U.S. Pat.No. 5,969,116, the disclosure of which is incorporated herein byreference in its entirety.

Some 2′ substituents may be cyclic compositions of the formula:

where L₁, L₂ and L₃ form a ring system having from about 4 to about 7carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2hetero atoms wherein said hetero atoms are oxygen, nitrogen or sulfurand wherein said ring system is aliphatic, unsaturated aliphatic,aromatic or heterocyclic;

R is OX, SX, N(H)X or NX₂;

X is H, C₁-C₈ alkyl, C₁-C₈haloalkyl, C(═NH)N(H)Z, C(═O)N(H)Z andOC(═O)N(H)Z;Y is alkyl or haloalkyl having 1 to about 10 carbon atoms, alkenylhaving 2 to about 10 carbon atoms, alkynyl having 2 to about 10 carbonatoms, aryl having 6 to about 14 carbon atoms, N(H)X, NX₂, OX, halo, SXor CN;n is 0, 1 or 2; andZ is H or C₁-C₈alkyl.

In certain embodiments, the ring system is phenyl, pyridyl, cyclopentyl,cyclobutyl, cyclohexyl, cycloheptyl, morpholino, piperidyl orpiperazinyl with the proviso that the elected ring system is mono-, di-,or tri-substituted. In other embodiments, R is OH, SH, OCH₃,N(H)C(═NH)NH₂, N(H)C(═O)NH₂, NH₂, or OC(═O)NH₂. In some embodiments, Liand L₂ are each carbon atoms. In yet other embodiments, Li and L₂ arecarbon atoms and the other of Li and L₂ is a heteroatom selected from O,S and N. These ligands may be made by methods taught by U.S. Pat. No.6,271,358, whose disclosure is incorporated herein by reference in itsentirety.

In some embodiments, the 2′ position of the sugar ring can have twosubstituents, Y1 and Y2; provided that both Y1 and Y2 are not H and thatwhen one of Y1 and Y2 is H and the other of Y1 and Y2 is OH, the sugarring is other than a ribose sugar. In certain embodiments, Y1 and Y2 areeach independently hydrogen; hydroxyl; halogen; C₂₋₄ alkenyl, C₂₋₄alkynyl, or C₁₋₄ alkyl optionally substituted with amino, hydroxy, or 1to 3 fluorine atoms; C₁₋₁₀ alkoxy, optionally substituted with C₁₋₃alkoxy, C₁₋₃ thioalkoxy or 1 to 3 fluorine atoms; C₂₋₆ alkenyloxy; C₁₋₄alkylthio; C₁₋₈ alkylcarbonyloxy; aryloxycarbonyl; azido; amino; C₁₋₄alkylamino; di(C₁₋₄ alkyl)amino; or Y³. Y³ is a conjugate molecule or areporter molecule. These substituents are described in more detail incommonly owned U.S. patent application Ser. No. 10/444,628, thedisclosure of which is incorporated by reference in its entirety. Insome preferred embodiments Y1 is C₂₋₄ alkenyl, C₂₋₄ alkynyl, or C₁₋₄alkyl, wherein the alkyl is unsubstituted or substituted with hydroxy,amino, C₁₋₄ alkoxy, C₁₋₄ alkylthio, or one to three fluorine atoms andY2 is hydrogen, fluorine, hydroxy, C₁₋₁₀ alkoxy, or C₁₋₁₀ alkyl. Inother preferred embodiments, Y1 is alkyl unsubstituted or substitutedwith hydroxy, amino, C₁₋₄ alkoxy, C₁₋₄ alkylthio, or one to threefluorine atoms, particularly where Y1 is methyl or trifluoromethyl. Inyet other preferred compounds, Y2 is hydrogen or hydroxyl. Thesesubstituents are described in commonly owned U.S. patent applicationSer. No. 10/444,298, the disclosure of which is incorporated herein byreference in its entirety.

Hybridization

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases that pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An oligomeric compound of the invention is believed to specificallyhybridize to the target nucleic acid and interfere with its normalfunction to cause a loss of activity. There is preferably a sufficientdegree of complementarity to avoid non-specific binding of theoligomeric compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the context of the present invention the phrase “stringenthybridization conditions” or “stringent conditions” refers to conditionsunder which an oligomeric compound of the invention will hybridize toits target sequence, but to a minimal number of other sequences.Stringent conditions are sequence-dependent and will vary with differentcircumstances and in the context of this invention; “stringentconditions” under which oligomeric compounds hybridize to a targetsequence are determined by the nature and composition of the oligomericcompounds and the assays in which they are being investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing of two nucleobases regardless of where the two are located. Forexample, if a nucleobase at a certain position of an oligomeric compoundis capable of hydrogen bonding with a nucleobase at a certain positionof a target nucleic acid, then the position of hydrogen bonding betweenthe oligomer and the target nucleic acid is considered to be acomplementary position. The oligomeric compound and the target nucleicacid are complementary to each other when a sufficient number ofcomplementary positions in each molecule are occupied by nucleobasesthat can hydrogen bond with each other. Thus, “specificallyhybridizable” and “complementary” are terms which are used to indicate asufficient degree of precise pairing or complementarity over asufficient number of nucleobases such that stable and specific bindingoccurs between the oligomer and a target nucleic acid.

It is understood in the art that the sequence of the oligomeric compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligomeric compound mayhybridize over one or more segments such that intervening or adjacentsegments are not involved in the hybridization event (e.g., a loopstructure or hairpin structure). It is preferred that the oligomericcompounds of the present invention comprise at least 70% sequencecomplementarity to a target region within the target nucleic acid, morepreferably that they comprise 90% sequence complementarity and even morepreferably comprise 95% sequence complementarity to the target regionwithin the target nucleic acid sequence to which they are targeted. Forexample, an oligomeric compound in which 18 of 20 nucleobases of theoligomeric compound are complementary to a target region, and wouldtherefore specifically hybridize, would represent 90 percentcomplementarity. In this example, the remaining noncomplementarynucleobases may be clustered or interspersed with complementarynucleobases and need not be contiguous to each other or to complementarynucleobases. As such, an oligomeric compound which is 18 nucleobases inlength having 4 (four) noncomplementary nucleobases which are flanked bytwo regions of complete complementarity with the target nucleic acidwould have 77.8% overall complementarity with the target nucleic acidand would thus fall within the scope of the present invention. Percentcomplementarity of an oligomeric compound with a region of a targetnucleic acid can be determined routinely using BLAST programs (basiclocal alignment search tools) and PowerBLAST programs known in the art(Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden,Genome Res., 1997, 7, 649-656).

Targets of the Invention

“Targeting” an oligomeric compound to a particular nucleic acidmolecule, in the context of this invention, can be a multistep process.The process usually begins with the identification of a target nucleicacid whose function is to be modulated. This target nucleic acid may be,for example, a mRNA transcribed from a cellular gene whose expression isassociated with a particular disorder or disease state, or a nucleicacid molecule from an infectious agent.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe interaction to occur such that the desired effect, e.g., modulationof expression, will result. Within the context of the present invention,the term “region” is defined as a portion of the target nucleic acidhaving at least one identifiable structure, function, or characteristic.Within regions of target nucleic acids are segments. “Segments” aredefined as smaller or sub-portions of regions within a target nucleicacid. “Sites,” as used in the present invention, are defined aspositions within a target nucleic acid. The terms region, segment, andsite can also be used to describe an oligomeric compound of theinvention such as for example a gapped oligomeric compound having 3separate segments.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding a nucleic acid target, regardless ofthe sequence(s) of such codons. It is also known in the art that atranslation termination codon (or “stop codon”) of a gene may have oneof three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the correspondingDNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense oligomeric compounds of thepresent invention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using oligomeric compounds targeted to, forexample, pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequences.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

The locations on the target nucleic acid to which preferred compoundsand compositions of the invention hybridize are herein below referred toas “preferred target segments.” As used herein the term “preferredtarget segment” is defined as at least an 8-nucleobase portion of atarget region to which an active antisense oligomeric compound istargeted. While not wishing to be bound by theory, it is presentlybelieved that these target segments represent portions of the targetnucleic acid that are accessible for hybridization.

Once one or more target regions, segments or sites have been identified,oligomeric compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

In accordance with an embodiment of the this invention, a series ofnucleic acid duplexes comprising the antisense strand oligomericcompounds of the present invention and their respective complement sensestrand compounds can be designed for a specific target or targets. Theends of the strands may be modified by the addition of one or morenatural or modified nucleobases to form an overhang. The sense strand ofthe duplex is designed and synthesized as the complement of theantisense strand and may also contain modifications or additions toeither terminus. For example, in one embodiment, both strands of theduplex would be complementary over the central nucleobases, each havingoverhangs at one or both termini.

For the purposes of describing an embodiment of this invention, thecombination of an antisense strand and a sense strand, each of can be ofa specified length, for example from 18 to 29 nucleotides long, isidentified as a complementary pair of siRNA oligomers. Thiscomplementary pair of siRNA oligomers can include additional nucleotideson either of their 5′ or 3′ ends. Further they can include othermolecules or molecular structures on their 3′ or 5′ ends such as aphosphate group on the 5′ end. A preferred group of compounds of theinvention include a phosphate group on the 5′ end of the antisensestrand compound. Other preferred compounds also include a phosphategroup on the 5′ end of the sense strand compound. An even furtherpreferred compounds would include additional nucleotides such as a twobase overhang on the 3′ end.

For example, a preferred siRNA complementary pair of oligomers comprisean antisense strand oligomeric compound having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 1) and having a two-nucleobase overhangof deoxythymidine(dT) and its complement sense strand. These oligomerswould have the following structure:

5′     c g a g a g g c g g a c g g g a c c g T T 3′ Antisense Strand(SEQ ID NO: 2)        | | | | | | | | | | | | | | | | | | | 3′T T g c t c t c c g c c t g c c c t g g c 5′ Complement Strand(SEQ ID NO: 3)

In an additional embodiment of the invention, a single oligomer havingboth the antisense portion as a first region in the oligomer and thesense portion as a second region in the oligomer is selected. The firstand second regions are linked together by either a nucleotide linker (astring of one or more nucleotides that are linked together in asequence) or by a non-nucleotide linker region or by a combination ofboth a nucleotide and non-nucleotide structure. In each of thesestructures, the oligomer, when folded back on itself, would becomplementary at least between the first region, the antisense portion,and the second region, the sense portion. Thus the oligomer would have apalindrome within it structure wherein the first region, the antisenseportion in the 5′ to 3′ direction, is complementary to the secondregion, the sense portion in the 3′ to 5′ direction.

In a further embodiment, the invention includes oligomer/proteincompositions. Such compositions have both an oligomer component and aprotein component. The oligomer component comprises at least oneoligomer, either the antisense or the sense oligomer but preferably theantisense oligomer (the oligomer that is antisense to the target nucleicacid). The oligomer component can also comprise both the antisense andthe sense strand oligomers. The protein component of the compositioncomprises at least one protein that forms a portion of the RNA-inducedsilencing complex, i.e., the RISC complex.

RISC is a ribonucleoprotein complex that contains an oligomer componentand proteins of the Argonaute family of proteins, among others. While wedo not wish to be bound by theory, the Argonaute proteins make up ahighly conserved family whose members have been implicated in RNAinterference and the regulation of related phenomena. Members of thisfamily have been shown to possess the canonical PAZ and Piwi domains,thought to be a region of protein-protein interaction. Other proteinscontaining these domains have been shown to effect target cleavage,including the RNAse, Dicer. The Argonaute family of proteins includes,but depending on species, are not necessary limited to, elF2C1 andelF2C2. elF2C2 is also known as human GERp95. While we do not wish to bebound by theory, at least the antisense oligomer strand is bound to theprotein component of the RISC complex. Additional, the complex mightalso include the sense strand oligomer. Carmell et al, Genes andDevelopment 2002, 16, 2733-2742.

Also while we do not wish to be bound by theory, it is further believedthat the RISC complex may interact with one or more of the translationmachinery components. Translation machinery components include but arenot limited to proteins that effect or aid in the translation of an RNAinto protein including the ribosomes or polyribosome complex. Therefore,in a further embodiment of the invention, the oligomer component of theinvention is associated with a RISC protein component and furtherassociates with the translation machinery of a cell. Such interactionwith the translation machinery of the cell would include interactionwith structural and enzymatic proteins of the translation machineryincluding but not limited to the polyribosome and ribosomal subunits.

In a further embodiment of the invention, the oligomer of the inventionis associated with cellular factors such as transporters or chaperones.These cellular factors can be protein, lipid or carbohydrate based andcan have structural or enzymatic functions that may or may not requirethe complexation of one or more metal ions.

Furthermore, the oligomer of the invention itself may have one or moremoieties which are bound to the oligomer which facilitate the active orpassive transport, localization or compartmentalization of the oligomer.Cellular localization includes, but is not limited to, localization towithin the nucleus, the nucleolus or the cytoplasm. Compartmentalizationincludes, but is not limited to, any directed movement of the oligomersof the invention to a cellular compartment including the nucleus,nucleolus, mitochondrion, or imbedding into a cellular membranesurrounding a compartment or the cell itself.

In a further embodiment of the invention, the oligomer of the inventionis associated with cellular factors that affect gene expression, morespecifically those involved in RNA modifications. These modificationsinclude, but are not limited to posttrascriptional modifications such asmethylation. Furthermore, the oligomer of the invention itself may haveone or more moieties which are bound to the oligomer which facilitatethe posttranscriptional modification.

The oligomeric compounds of the invention may be used in the form ofsingle-stranded, double-stranded, circular or hairpin oligomericcompounds and may contain structural elements such as internal orterminal bulges or loops. Once introduced to a system, the oligomericcompounds of the invention may interact with or elicit the action of oneor more enzymes or may interact with one or more structural proteins toeffect modification of the target nucleic acid.

One non-limiting example of such an interaction is the RISC complex. Useof the RISC complex to effect cleavage of RNA targets thereby greatlyenhances the efficiency of oligomer-mediated inhibition of geneexpression. Similar roles have been postulated for other ribonucleasessuch as those in the RNase III and ribonuclease L family of enzymes.

Preferred forms of oligomeric compound of the invention include asingle-stranded antisense oligomer that binds in a RISC complex, adouble stranded antisense/sense pair of oligomer or a single strandoligomer that includes both an antisense portion and a sense portion.Each of these compounds or compositions is used to induce potent andspecific modulation of gene function.

Such specific modulation of gene function has been shown in many speciesby the introduction of double-stranded structures, such asdouble-stranded RNA (dsRNA) molecules and has been shown to inducepotent and specific antisense-mediated reduction of the function of agene or its associated gene products. This phenomenon occurs in bothplants and animals and is believed to have an evolutionary connection toviral defense and transposon silencing.

The compounds and compositions of the invention are used to modulate theexpression of a target nucleic acid. “Modulators” are those oligomericcompounds that decrease or increase the expression of a nucleic acidmolecule encoding a target and which comprise at least an 8-nucleobaseportion that is complementary to a preferred target segment. Thescreening method comprises the steps of contacting a preferred targetsegment of a nucleic acid molecule encoding a target with one or morecandidate modulators, and selecting for one or more candidate modulatorswhich decrease or increase the expression of a nucleic acid moleculeencoding a target. Once it is shown that the candidate modulator ormodulators are capable of modulating (e.g. either decreasing orincreasing) the expression of a nucleic acid molecule encoding a target,the modulator may then be employed in further investigative studies ofthe function of a target, or for use as a research, diagnostic, ortherapeutic agent in accordance with the present invention.

Oligomeric Compounds

In the context of the present invention, the term “oligomeric compound”refers to a polymeric structure capable of hybridizing a region of anucleic acid molecule. This term includes oligonucleotides,oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics andcombinations of these. Oligomeric compounds routinely prepared linearlybut can be joined or otherwise prepared to be circular and may alsoinclude branching. Oligomeric compounds can hybridized to form doublestranded compounds that can be blunt ended or may include overhangs. Ingeneral an oligomeric compound comprises a backbone of linked momericsubunits where each linked momeric subunit is directly or indirectlyattached to a heterocyclic base moiety. The linkages joining themonomeric subunits, the sugar moieties or surrogates and theheterocyclic base moieties can be independently modified giving rise toa plurality of motifs for the resulting oligomeric compounds includinghemimers, gapmers and chimeras.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base moiety.The two most common classes of such heterocyclic bases are purines andpyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligomers, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. The respective ends of this linear polymericstructure can be joined to form a circular structure by hybridization orby formation of a covalent bond, however, open linear structures aregenerally preferred. Within the oligomer structure, the phosphate groupsare commonly referred to as forming the intemucleoside linkages of theoligomer. The normal internucleoside linkage of RNA and DNA is a 3′ to5′ phosphodiester linkage.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA). This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent intemucleosidelinkages. The term “oligonucleotide analog” refers to oligonucleotidesthat have one or more non-naturally occurring portions which function ina similar manner to oligonulceotides. Such non-naturally occurringoligonucleotides are often preferred the naturally occurring formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

In the context of this invention, the term “oligonucleoside” refers tonucleosides that are joined by internucleoside linkages that do not havephosphorus atoms. Internucleoside linkages of this type include shortchain alkyl, cycloalkyl, mixed heteroatom alkyl, mixed heteroatomcycloalkyl, one or more short chain heteroatomic and one or more shortchain heterocyclic. These intemucleoside linkages include but are notlimited to siloxane, sulfide, sulfoxide, sulfone, acetal, formacetal,thioformacetal, methylene formacetal, thioformacetal, alkeneyl,sulfamate; methyleneimino, methylenehydrazino, sulfonate, sulfonamide,amide and others having mixed N, O, S and CH₂ component parts.

In addition to the modifications described above, the nucleosides of theoligomeric compounds of the invention can have a variety of othermodification so long as these other modifications either alone or incombination with other nucleosides enhance one or more of the desiredproperties described above. Thus, for nucleotides that are incorporatedinto oligomers of the invention, these nucleotides can have sugarportions that correspond to naturally-occurring sugars or modifiedsugars. Representative modified sugars include carbocyclic or acyclicsugars, sugars having substituent groups at one or more of their 2′, 3′or 4′ positions and sugars having substituents in place of one or morehydrogen atoms of the sugar. Additional nucleosides amenable to thepresent invention having altered base moieties and or altered sugarmoieties are disclosed in U.S. Pat. No. 3,687,808 and PCT applicationPCT/US89/02323.

Altered base moieties or altered sugar moieties also include othermodifications consistent with the spirit of this invention. Sucholigomers are best described as being structurally distinguishable from,yet functionally interchangeable with, naturally occurring or syntheticwild type oligonucleotides. All such oligomers are comprehended by thisinvention so long as they function effectively to mimic the structure ofa desired RNA or DNA strand. A class of representative basemodifications include tricyclic cytosine analog, termed “G clamp” (Lin,et al., J. Am. Chem. Soc. 1998, 120, 8531). This analog makes fourhydrogen bonds to a complementary guanine (G) within a helix bysimultaneously recognizing the Watson-Crick and Hoogsteen faces of thetargeted G. This G clamp modification when incorporated intophosphorothioate oligomers, dramatically enhances antisense potencies incell culture. The oligomers of the invention also can includephenoxazine-substituted bases of the type disclosed by Flanagan, et al.,Nat. Biotechnol. 1999, 17(1), 48-52.

The oligomeric compounds in accordance with this invention preferablycomprise from about 8 to about 80 nucleobases (i.e. from about 8 toabout 80 linked nucleosides). One of ordinary skill in the art willappreciate that the invention embodies oligomeric compounds of 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80nucleobases in length.

In one preferred embodiment, the oligomeric compounds of the inventionare 12 to 50 nucleobases in length. One having ordinary skill in the artwill appreciate that this embodies oligomeric compounds of 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 nucleobases in length.

In another preferred embodiment, the oligomeric compounds of theinvention are 15 to 30 nucleobases in length. One having ordinary skillin the art will appreciate that this embodies oligomeric compounds of15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleobases in length.

Particularly preferred oligomeric compounds are oligomers from about 12to about 50 nucleobases, even more preferably those comprising fromabout 15 to about 30 nucleobases.

General Oligomer Synthesis

Oligomerization of modified and unmodified nucleosides is performedaccording to literature procedures for DNA like compounds (Protocols forOligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press) and/orRNA like compounds (Scaringe, Methods (2001), 23, 206-217. Gait et al.,Applications of Chemically synthesized RNA in RNA:Protein Interactions,Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron (2001), 57, 5707-5713)synthesis as appropriate. In addition specific protocols for thesynthesis of oligomeric compounds of the invention are illustrated inthe examples below.

RNA oligomers can be synthesized by methods disclosed herein orpurchased from various RNA synthesis companies such as for exampleDharmacon Research Inc., (Lafayette, Colo.).

Irrespective of the particular protocol used, the oligomeric compoundsused in accordance with this invention may be conveniently and routinelymade through the well-known technique of solid phase synthesis.Equipment for such synthesis is sold by several vendors including, forexample, Applied Biosystems (Foster City, Calif.). Any other means forsuch synthesis known in the art may additionally or alternatively beemployed.

For double stranded structures of the invention, once synthesized, thecomplementary strands preferably are annealed. The single strands arealiquoted and diluted to a concentration of 50 uM. Once diluted, 30 uLof each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of the buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA compound is 20 uM. This solutioncan be stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the desired synthetic duplexes are evaluated for theirability to modulate target expression. When cells reach 80% confluency,they are treated with synthetic duplexes comprising at least oneoligomeric compound of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired dsRNA compound at a finalconcentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Oligomer and Monomer Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligomers, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligomers, the phosphate groups are commonly referredto as forming the internucleoside linkage or in conjunction with thesugar ring the backbone of the oligomer. The normal internucleosidelinkage that makes up the backbone of RNA and DNA is a 3′ to 5′phosphodiester linkage.

Modified Internucleoside Linkages

Specific examples of preferred antisense oligomeric compounds useful inthis invention include oligomers containing modified e.g. non-naturallyoccurring internucleoside linkages. As defined in this specification,oligomers having modified internucleoside linkages includeinternucleoside linkages that retain a phosphorus atom andinternucleoside linkages that do not have a phosphorus atom. For thepurposes of this specification, and as sometimes referenced in the art,modified oligomers that do not have a phosphorus atom in theirinternucleoside backbone can also be considered to be oligonucleosides.

In the C. elegans system, modification of the internucleotide linkage(phosphorothioate) did not significantly interfere with RNAi activity.Based on this observation, it is suggested that certain preferredoligomeric compounds of the invention can also have one or more modifiedinternucleoside linkages. A preferred phosphorus containing modifiedinternucleoside linkage is the phosphorothioate internucleoside linkage.

Preferred modified oligomer backbones containing a phosphorus atomtherein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligomers having inverted polarity comprise a single 3′ to 3′linkage at the 3′-most internucleotide linkage i.e. a single invertednucleoside residue which may be abasic (the nucleobase is missing or hasa hydroxyl group in place thereof). Various salts, mixed salts and freeacid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

In more preferred embodiments of the invention, oligomeric compoundshave one or more phosphorothioate and/or heteroatom internucleosidelinkages, in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as amethylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester internucleotide linkage is represented as—O—P(═O)(OH)—O—CH₂—]. The MMI type internucleoside linkages aredisclosed in the above referenced U.S. Pat. No. 5,489,677. Preferredamide internucleoside linkages are disclosed in the above referencedU.S. Pat. No. 5,602,240.

Preferred modified oligomer backbones that do not include a phosphorusatom therein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetal and thioformacetal backbones; methylene formacetaland thioformacetal backbones; riboacetal backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S.: 5,034,506;5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562;5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677;5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240;5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which arecommonly owned with this application, and each of which is hereinincorporated by reference.

Oligomer Mimetics

Another preferred group of oligomeric compounds amenable to the presentinvention includes oligonucleotide mimetics. The term mimetic as it isapplied to oligonucleotides is intended to include oligomeric compoundswherein only the furanose ring or both the furanose ring and theinternucleotide linkage are replaced with novel groups, replacement ofonly the furanose ring is also referred to in the art as being a sugarsurrogate. The heterocyclic base moiety or a modified heterocyclic basemoiety is maintained for hybridization with an appropriate targetnucleic acid. One such oligomeric compound, an oligonucleotide mimeticthat has been shown to have excellent hybridization properties, isreferred to as a peptide nucleic acid (PNA). In PNA oligomericcompounds, the sugar-backbone of an oligonucleotide is replaced with anamide containing backbone, in particular an aminoethylglycine backbone.The nucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. RepresentativeUnited States patents that teach the preparation of PNA oligomericcompounds include, but are not limited to, U.S.: 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA oligomeric compounds can be found in Nielsen etal., Science, 1991, 254, 1497-1500.

One oligonucleotide mimetic that has been reported to have excellenthybridization properties is peptide nucleic acids (PNA). The backbone inPNA compounds is two or more linked aminoethylglycine units which givesPNA an amide containing backbone. The heterocyclic base moieties arebound directly or indirectly to aza nitrogen atoms of the amide portionof the backbone.

Representative United States patents that teach the preparation of PNAcompounds include, but are not limited to, U.S.: 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500.

PNA has been modified to incorporate numerous modifications since thebasic PNA structure was first prepared. The basic structure is shownbelow:

wherein

Bx is a heterocyclic base moiety;

T₄ is hydrogen, an amino protecting group, —C(O)R₅, substituted orunsubstituted C₁-C₁₀ alkyl, substituted or unsubstituted C₂-C₁₀ alkenyl,substituted or unsubstituted C₂-C₁₀ alkynyl, alkylsulfonyl,arylsulfonyl, a chemical functional group, a reporter group, a conjugategroup, a D or L α-amino acid linked via the α-carboxyl group oroptionally through the co-carboxyl group when the amino acid is asparticacid or glutamic acid or a peptide derived from D, L or mixed D and Lamino acids linked through a carboxyl group, wherein the substituentgroups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl;

T₅ is —OH, —N(Z₁)Z₂, R₅, D or L α-amino acid linked via the α-aminogroup or optionally through the co-amino group when the amino acid islysine or ornithine or a peptide derived from D, L or mixed D and Lamino acids linked through an amino group, a chemical functional group,a reporter group or a conjugate group;

Z₁ is hydrogen, C₁-C₆ alkyl, or an amino protecting group;

Z₂ is hydrogen, C₁-C₆ alkyl, an amino protecting group,—C(═O)—(CH₂)_(n)-J-Z₃, a D or L α-amino acid linked via the α-carboxylgroup or optionally through the co-carboxyl group when the amino acid isaspartic acid or glutamic acid or a peptide derived from D, L or mixed Dand L amino acids linked through a carboxyl group;

Z₃ is hydrogen, an amino protecting group, —C₁-C₆ alkyl, —C(═O)—CH₃,benzyl, benzoyl, or —(CH₂)_(n)—N(H)Z₁;

each J is O, S or NH;

R₅ is a carbonyl protecting group; and

n is from 2 to about 50.

Another class of oligonucleotide mimetic that has been studied is basedon linked morpholino units (morpholino nucleic acid) having heterocyclicbases attached to the morpholino ring. A number of linking groups havebeen reported that link the morpholino monomeric units in a morpholinonucleic acid. A preferred class of linking groups have been selected togive a non-ionic oligomeric compound. The non-ionic morpholino-basedoligomeric compounds are less likely to have undesired interactions withcellular proteins. Morpholino-based oligomeric compounds are non-ionicmimics of oligonucleotides which are less likely to form undesiredinteractions with cellular proteins (Dwaine A. Braasch and David R.Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-basedoligomeric compounds are disclosed in U.S. Pat. No. 5,034,506, issuedJul. 23, 1991. The morpholino class of oligomeric compounds have beenprepared having a variety of different linking groups joining themonomeric subunits.

Morpholino nucleic acids have been prepared having a variety ofdifferent linking groups (L₂) joining the monomeric subunits. The basicformula is shown below:

wherein

T₁ is hydroxyl or a protected hydroxyl;

T₅ is hydrogen or a phosphate or phosphate derivative;

L₂ is a linking group; and

n is from 2 to about 50.

A further class of oligonucleotide mimetic is referred to ascyclohexenyl nucleic acids (CeNA). The furanose ring normally present inan DNA/RNA molecule is replaced with a cyclohenyl ring. CeNA DMTprotected phosphoramidite monomers have been prepared and used foroligomeric compound synthesis following classical phosphoramiditechemistry. Fully modified CeNA oligomeric compounds and oligomers havingspecific positions modified with CeNA have been prepared and studied(see Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602). In generalthe incorporation of CeNA monomers into a DNA chain increases itsstability of a DNA/RNA hybrid. CeNA oligoadenylates formed complexeswith RNA and DNA complements with similar stability to the nativecomplexes. The study of incorporating CeNA structures into naturalnucleic acid structures was shown by NMR and circular dichroism toproceed with easy conformational adaptation. Furthermore theincorporation of CeNA into a sequence targeting RNA was stable to serumand able to activate E. Coli RNase resulting in cleavage of the targetRNA strand.

The general formula of CeNA is shown below:

wherein

each Bx is a heterocyclic base moiety;

T₁ is hydroxyl or a protected hydroxyl; and

T₂ is hydroxyl or a protected hydroxyl.

Another preferred group of oligomeric compounds amenable to the presentinvention includes oligonucleotide mimetics. The term mimetic as it isapplied to oligomers is intended to include oligomeric compounds whereinonly the furanose ring or both the furanose ring and the internucleotidelinkage are replaced with novel groups, replacement of only the furanosering is also referred to in the art as being a sugar surrogate. Theheterocyclic base moiety or a modified heterocyclic base moiety ismaintained for hybridization with an appropriate target nucleic acid.

Another class of oligonucleotide mimetic (anhydrohexitol nucleic acid)can be prepared from one or more anhydrohexitol nucleosides (see,Wouters and Herdewijn, Bioorg. Med. Chem. Lett., 1999, 9, 1563-1566) andwould have the general formula:

A further preferred modification includes Locked Nucleic Acids (LNAs) inwhich the 2′-hydroxyl group is linked to the 4′ carbon atom of the sugarring thereby forming a 2′-C,4′-C-oxymethylene linkage thereby forming abicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_(n)group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1or 2 (Singh et al., Chem. Commun., 1998, 4, 455-456). LNA and LNAanalogs display very high duplex thermal stabilities with complementaryDNA and RNA (Tm=+3 to +10 C), stability towards 3′-exonucleolyticdegradation and good solubility properties. The basic structure of LNAshowing the bicyclic ring system is shown below:

The conformations of LNAs determined by 2D NMR spectroscopy have shownthat the locked orientation of the LNA nucleotides, both insingle-stranded LNA and in duplexes, constrains the phosphate backbonein such a way as to introduce a higher population of the N-typeconformation (Petersen et al., J. Mol. Recognit., 2000, 13, 44-53).These conformations are associated with improved stacking of thenucleobases (Wengel et al., Nucleosides Nucleotides, 1999, 18,1365-1370).

LNA has been shown to form exceedingly stable LNA:LNA duplexes (Koshkinet al., J. Am. Chem. Soc., 1998, 120, 13252-13253). LNA:LNAhybridization was shown to be the most thermally stable nucleic acidtype duplex system, and the RNA-mimicking character of LNA wasestablished at the duplex level. Introduction of 3 LNA monomers (T or A)significantly increased melting points (Tm=+15/+11) toward DNAcomplements. The universality of LNA-mediated hybridization has beenstressed by the formation of exceedingly stable LNA:LNA duplexes. TheRNA-mimicking of LNA was reflected with regard to the N-typeconformational restriction of the monomers and to the secondarystructure of the LNA:RNA duplex.

LNAs also form duplexes with complementary DNA, RNA or LNA with highthermal affinities. Circular dichroism (CD) spectra show that duplexesinvolving fully modified LNA (esp. LNA:RNA) structurally resemble anA-form RNA:RNA duplex. Nuclear magnetic resonance (NMR) examination ofan LNA:DNA duplex confirmed the 3′-endo conformation of an LNA monomer.Recognition of double-stranded DNA has also been demonstrated suggestingstrand invasion by LNA. Studies of mismatched sequences show that LNAsobey the Watson-Crick base pairing rules with generally improvedselectivity compared to the corresponding unmodified reference strands.Novel types of LNA-oligomeric compounds, as well as the LNAs, are usefulin a wide range of diagnostic and therapeutic applications. Among theseare antisense applications, PCR applications, strand-displacementoligomers, substrates for nucleic acid polymerases and generally asnucleotide based drugs.

Potent and nontoxic antisense oligomers containing LNAs have beendescribed (Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A, 2000, 97,5633-5638.) The authors have demonstrated that LNAs confer severaldesired properties to antisense agents. LNA/DNA copolymers were notdegraded readily in blood serum and cell extracts. LNA/DNA copolymersexhibited potent antisense activity in assay systems as disparate asG-protein-coupled receptor signaling in living rat brain and detectionof reporter genes in Escherichia coli. Lipofectin-mediated efficientdelivery of LNA into living human breast cancer cells has also beenaccomplished.

The synthesis and preparation of the LNA monomers adenine, cytosine,guanine, 5-methyl-cytosine, thymine and uracil, along with theiroligomerization, and nucleic acid recognition properties have beendescribed (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). LNAs andpreparation thereof are also described in WO 98/39352 and WO 99/14226.

The first analogs of LNA, phosphorothioate-LNA and 2′-thio-LNAs, havealso been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8,2219-2222). Preparation of locked nucleoside analogs containingoligodeoxyribonucleotide duplexes as substrates for nucleic acidpolymerases has also been described (Wengel et al., PCT InternationalApplication WO 98-DK393 19980914). Furthermore, synthesis of2′-amino-LNA, a novel conformationally restricted high-affinityoligonucleotide analog with a handle has been described in the art(Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition,2′-Amino- and 2′-methylamino-LNA's have been prepared and the thermalstability of their duplexes with complementary RNA and DNA strands hasbeen previously reported.

Further oligonucleotide mimetics have been prepared to include bicyclicand tricyclic nucleoside analogs having the formulas (amidite monomersshown):

(see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439; Steffens etal., J. Am. Chem. Soc., 1999, 121, 3249-3255; and Renneberg et al., JAm. Chem. Soc., 2002, 124, 5993-6002). These modified nucleoside analogshave been oligomerized using the phosphoramidite approach and theresulting oligomeric compounds containing tricyclic nucleoside analogshave shown increased thermal stabilities (Tm's) when hybridized to DNA,RNA and itself. Oligomeric compounds containing bicyclic nucleosideanalogs have shown thermal stabilities approaching that of DNA duplexes.

Another class of oligonucleotide mimetic is referred to asphosphonomonoester nucleic acids incorporate a phosphorus group in abackbone the backbone. This class of olignucleotide mimetic is reportedto have useful physical and biological and pharmacological properties inthe areas of inhibiting gene expression (antisense oligonucleotides,ribozymes, sense oligonucleotides and triplex-forming oligonucleotides),as probes for the detection of nucleic acids and as auxiliaries for usein molecular biology.

The general formula (for definitions of variables see: U.S. Pat. Nos.5,874,553 and 6,127,346 herein incorporated by reference in theirentirety) is shown below.

Another oligonucleotide mimetic has been reported wherein the furanosylring has been replaced by a cyclobutyl moiety.

Modified Nucleobases Naturally Occurring Nucleobases

Oligomeric compounds may also include nucleobase (often referred to inthe art simply as “base” or “heterocyclic base moiety”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesalso referred herein as heterocyclic base moieties include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl(—C≡C—CH₃) uracil and cytosine and other alkynyl derivatives ofpyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl and other 8-substituted adenines and guanines, 5-haloparticularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

Heterocyclic base moieties may also include those in which the purine orpyrimidine base is replaced with other heterocycles, for example7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds., Antisense Research andApplications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications.

In one aspect of the present invention oligomeric compounds are preparedhaving polycyclic heterocyclic compounds in place of one or moreheterocyclic base moieties. A number of tricyclic heterocyclic compoundshave been previously reported. These compounds are routinely used inantisense applications to increase the binding properties of themodified strand to a target strand. The most studied modifications aretargeted to guanosines hence they have been termed G-clamps or cytidineanalogs. Many of these polycyclic heterocyclic compounds have thegeneral formula:

Representative cytosine analogs that make 3 hydrogen bonds with aguanosine in a second strand include 1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═H) [Kurchavov, et al., Nucleosides and Nucleotides, 1997, 16,1837-1846], 1,3-diazaphenothiazine-2-one (R₁₀═S, R₁₁-R₁₄═H), [Lin,K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117,3873-3874] and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R₁₀═O,R₁₁-R₁₄═F) [Wang, J.; Lin, K.-Y., Matteucci, M. Tetrahedron Lett. 1998,39, 8385-8388]. Incorporated into oligonucleotides these basemodifications were shown to hybridize with complementary guanine and thelatter was also shown to hybridize with adenine and to enhance helicalthermal stability by extended stacking interactions (also see U.S.patent application entitled “Modified Peptide Nucleic Acids” filed May24, 2002, Ser. No. 10/155,920; and U.S. patent application entitled“Nuclease Resistant Chimeric Oligonucleotides” filed May 24, 2002, Ser.No. 10/013,295, both of which are commonly owned with this applicationand are herein incorporated by reference in their entirety).

Further helix-stabilizing properties have been observed when a cytosineanalog/substitute has an aminoethoxy moiety attached to the rigid1,3-diazaphenoxazine-2-one scaffold (R₁₀—O, R₁₁═—O—(CH₂)₂—NH₂, R₁₂₋₁₄═H)[Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532].Binding studies demonstrated that a single incorporation could enhancethe binding affinity of a model oligonucleotide to its complementarytarget DNA or RNA with a ΔT_(m) of up to 18° relative to 5-methylcytosine (dC5^(me)), which is the highest known affinity enhancement fora single modification, yet. On the other hand, the gain in helicalstability does not compromise the specificity of the oligomers. The Tmdata indicate an even greater discrimination between the perfect matchand mismatched sequences compared to dC5^(me). It was suggested that thetethered amino group serves as an additional hydrogen bond donor tointeract with the Hoogsteen face, namely the O6, of a complementaryguanine thereby forming 4 hydrogen bonds. This means that the increasedaffinity of G-clamp is mediated by the combination of extended basestacking and additional specific hydrogen bonding.

Further tricyclic heterocyclic compounds and methods of using them thatare amenable to the present invention are disclosed in U.S. Pat. No.6,028,183, which issued on May 22, 2000, and U.S. Pat. No. 6,007,992,which issued on Dec. 28, 1999, the contents of both are commonlyassigned with this application and are incorporated herein in theirentirety.

The enhanced binding affinity of the phenoxazine derivatives togetherwith their uncompromised sequence specificity make them valuablenucleobase analogs for the development of more potent antisense-baseddrugs. In fact, promising data have been derived from in vitroexperiments demonstrating that heptanucleotides containing phenoxazinesubstitutions are capable to activate RNaseH, enhance cellular uptakeand exhibit an increased antisense activity [Lin, K-Y; Matteucci, M. J.Am. Chem. Soc. 1998, 120, 8531-8532]. The activity enhancement was evenmore pronounced in case of G-clamp, as a single substitution was shownto significantly improve the in vitro potency of a 20mer2′-deoxyphosphorothioate oligonucleotides [Flanagan, W. M.; Wolf, J. J.;Olson, P.; Grant, D.; Lin, K.-Y.; Wagner, R. W.; Matteucci, M. Proc.Natl. Acad. Sci. USA, 1999, 96, 3513-3518]. Nevertheless, to optimizeoligomer design and to better understand the impact of theseheterocyclic modifications on the biological activity, it is importantto evaluate their effect on the nuclease stability of the oligomers.

Further modified polycyclic heterocyclic compounds useful asheterocyclic bases are disclosed in but not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257; 5,457,187;5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269; 5,750,692;5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S. patentapplication Ser. No. 09/996,292 filed Nov. 28, 2001, certain of whichare commonly owned with the instant application, and each of which isherein incorporated by reference.

Conjugates

A further preferred substitution that can be appended to the oligomericcompounds of the invention involves the linkage of one or more moietiesor conjugates which enhance the activity, cellular distribution orcellular uptake of the resulting oligomeric compounds. In one embodimentsuch modified oligomeric compounds are prepared by covalently attachingconjugate groups to functional groups such as hydroxyl or amino groups.Conjugate groups of the invention include intercalators, reportermolecules, polyamines, polyamides, polyethylene glycols, polyethers,groups that enhance the pharmacodynamic properties of oligomers, andgroups that enhance the pharmaco-kinetic properties of oligomers.Typical conjugates groups include cholesterols, lipids, phospho-lipids,biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance thepharmacodynamic properties, in the context of this invention, includegroups that improve oligomer uptake, enhance oligomer resistance todegradation, and/or strengthen sequence-specific hybridization with RNA.Groups that enhance the pharmacokinetic properties, in the context ofthis invention, include groups that improve oligomer uptake,distribution, metabolism or excretion. Representative conjugate groupsare disclosed in International Patent Application PCT/US92/09196, filedOct. 23, 1992 the entire disclosure of which is incorporated herein byreference.

Conjugate moieties include but are not limited to lipid moieties such asa cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharanet al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937.

The oligomeric compounds of the invention may also be conjugated toactive drug substances, for example, aspirin, warfarin, phenylbutazone,ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen,carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indomethicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drugconjugates and their preparation are described in U.S. patentapplication Ser. No. 09/334,130 (filed Jun. 15, 1999) which isincorporated herein by reference in its entirety.

Representative United States patents that teach the preparation of sucholigomer conjugates include, but are not limited to, U.S.: 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802;5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941;4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963;5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469;5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241,5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785;5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726;5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference.

Chimeric Oligomeric Compounds

It is not necessary for all positions in an oligomeric compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligomeric compound oreven at a single monomeric subunit such as a nucleoside within aoligomeric compound. The present invention also includes oligomericcompounds which are chimeric oligomeric compounds. “Chimeric” oligomericcompounds or “chimeras,” in the context of this invention, areoligomeric compounds that contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a nucleic acid based oligomer.

Chimeric oligomeric compounds typically contain at least one regionmodified so as to confer increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the oligomeric compound mayserve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNAhybrids. By way of example, RNase H is a cellular endonuclease whichcleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H,therefore, results in cleavage of the RNA target, thereby greatlyenhancing the efficiency of inhibition of gene expression. Consequently,comparable results can often be obtained with shorter oligomericcompounds when chimeras are used, compared to for examplephosphorothioate deoxyoligonucleotides hybridizing to the same targetregion. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

Chimeric oligomeric compounds of the invention may be formed ascomposite structures of two or more oligonucleotides, oligonucleotideanalogs, oligonucleosides and/or oligonucleotide mimetics as describedabove. Such oligomeric compounds have also been referred to in the artas hybrids hemimers, gapmers or inverted gapmers. Representative UnitedStates patents that teach the preparation of such hybrid structuresinclude, but are not limited to, U.S.: 5,013,830; 5,149,797; 5,220,007;5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;5,652,355; 5,652,356; and 5,700,922, certain of which are commonly ownedwith the instant application, and each of which is herein incorporatedby reference in its entirety.

3′-Endo Modifications

In one aspect of the present invention oligomeric compounds includenucleosides synthetically modified to induce a 3′-endo sugarconformation. A nucleoside can incorporate synthetic modifications ofthe heterocyclic base, the sugar moiety or both to induce a desired3′-endo sugar conformation. These modified nucleosides are used to mimicRNA like nucleosides so that particular properties of an oligomericcompound can be enhanced while maintaining the desirable 3′-endoconformational geometry. There is an apparent preference for an RNA typeduplex (A form helix, predominantly 3′-endo) as a requirement (e.g.trigger) of RNA interference which is supported in part by the fact thatduplexes composed of 2′-deoxy-2′-F-nucleosides appears efficient intriggering RNAi response in the C. elegans system. Properties that areenhanced by using more stable 3′-endo nucleosides include but aren'tlimited to modulation of pharmacokinetic properties through modificationof protein binding, protein off-rate, absorption and clearance;modulation of nuclease stability as well as chemical stability;modulation of the binding affinity and specificity of the oligomer(affinity and specificity for enzymes as well as for complementarysequences); and increasing efficacy of RNA cleavage. The presentinvention provides oligomeric triggers of RNAi having one or morenucleosides modified in such a way as to favor a C3′-endo typeconformation.

Nucleoside conformation is influenced by various factors includingsubstitution at the 2′, 3′ or 4′-positions of the pentofuranosyl sugar.Electronegative substituents generally prefer the axial positions, whilesterically demanding substituents generally prefer the equatorialpositions (Principles of Nucleic Acid Structure, Wolfgang Sanger, 1984,Springer-Verlag.) Modification of the 2′ position to favor the 3′-endoconformation can be achieved while maintaining the 2′-OH as arecognition element (Gallo et al., Tetrahedron (2001), 57, 5707-5713.Harry-O'kuru et al., J. Org. Chem., (1997), 62(6), 1754-1759 and Tang etal., J. Org. Chem. (1999), 64, 747-754.) Alternatively, preference forthe 3′-endo conformation can be achieved by deletion of the 2′-OH asexemplified by 2′ deoxy-2′F-nucleosides (Kawasaki et al., J. Med. Chem.(1993), 36, 831-841), which adopts the 3′-endo conformation positioningthe electronegative fluorine atom in the axial position. Othermodifications of the ribose ring, for example substitution at the4′-position to give 4′-F modified nucleosides (Guillerm et al.,Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and Owenet al., J. Org. Chem. (1976), 41, 3010-3017), or for examplemodification to yield methanocarba nucleoside analogs (Jacobson et al.,J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al., Bioorganic andMedicinal Chemistry Letters (2001), 11, 1333-1337) also inducepreference for the 3′-endo conformation. Along similar lines, oligomerictriggers of RNAi response might be composed of one or more nucleosidesmodified in such a way that conformation is locked into a C3′-endo typeconformation, i.e. Locked Nucleic Acid (LNA, Singh et al, Chem. Commun.(1998), 4, 455-456), and ethylene bridged Nucleic Acids (ENA, Morita etal, Bioorganic & Medicinal Chemistry Letters (2002), 12, 73-76.)Examples of modified nucleosides amenable to the present invention areshown below in

TABLE I These examples are meant to be representative and notexhaustive.

The preferred conformation of modified nucleosides and their oligomerscan be estimated by various methods such as molecular dynamicscalculations, nuclear magnetic resonance spectroscopy and CDmeasurements. Hence, modifications predicted to induce RNA likeconformations, A-form duplex geometry in an oligomeric context, areselected for use in the modified oligomers of the present invention. Thesynthesis of numerous of the modified nucleosides amenable to thepresent invention are known in the art (see for example, Chemistry ofNucleosides and Nucleotides Vol 1-3, ed. Leroy B. Townsend, 1988, Plenumpress., and the examples section below.) Nucleosides known to beinhibitors/substrates for RNA dependent RNA polymerases (for example HCVNS5B

In one aspect, the present invention is directed to oligomers that areprepared having enhanced properties compared to native RNA againstnucleic acid targets. A target is identified and an oligomer is selectedhaving an effective length and sequence that is complementary to aportion of the target sequence. Each nucleoside of the selected sequenceis scrutinized for possible enhancing modifications. A preferredmodification would be the replacement of one or more RNA nucleosideswith nucleosides that have the same 3′-endo conformational geometry.Such modifications can enhance chemical and nuclease stability relativeto native RNA while at the same time being much cheaper and easier tosynthesize and/or incorporate into an oligomer. The selected sequencecan be further divided into regions and the nucleosides of each regionevaluated for enhancing modifications that can be the result of achimeric configuration. Consideration is also given to the 5′ and3′-termini as there are often advantageous modifications that can bemade to one or more of the terminal nucleosides. The oligomericcompounds of the present invention include at least one 5′-modifiedphosphate group on a single strand or on at least one 5′-position of adouble stranded sequence or sequences. Further modifications are alsoconsidered such as internucleoside linkages, conjugate groups,substitute sugars or bases, substitution of one or more nucleosides withnucleoside mimetics and any other modification that can enhance theselected sequence for its intended target.

The terms used to describe the conformational geometry of homoduplexnucleic acids are “A Form” for RNA and “B Form” for DNA. The respectiveconformational geometry for RNA and DNA duplexes was determined fromX-ray diffraction analysis of nucleic acid fibers (Arnott and Hukins,Biochem. Biophys. Res. Comm., 1970, 47, 1504.) In general, RNA:RNAduplexes are more stable and have higher melting temperatures (Tm's)than DNA:DNA duplexes (Sanger et al., Principles of Nucleic AcidStructure, 1984, Springer-Verlag; New York, N.Y.; Lesnik et al.,Biochemistry, 1995, 34, 10807-10815; Conte et al., Nucleic Acids Res.,1997, 25, 2627-2634). The increased stability of RNA has been attributedto several structural features, most notably the improved base stackinginteractions that result from an A-form geometry (Searle et al., NucleicAcids Res., 1993, 21, 2051-2056). The presence of the 2′ hydroxyl in RNAbiases the sugar toward a C3′ endo pucker, i.e., also designated asNorthern pucker, which causes the duplex to favor the A-form geometry.In addition, the 2′ hydroxyl groups of RNA can form a network of watermediated hydrogen bonds that help stabilize the RNA duplex (Egli et al.,Biochemistry, 1996, 35, 8489-8494). On the other hand, deoxy nucleicacids prefer a C2′ endo sugar pucker, i.e., also known as Southernpucker, which is thought to impart a less stable B-form geometry(Sanger, W. (1984) Principles of Nucleic Acid Structure,Springer-Verlag, New York, N.Y.). As used herein, B-form geometry isinclusive of both C2′-endo pucker and 04′-endo pucker. This isconsistent with Berger, et. al., Nucleic Acids Research, 1998, 26,2473-2480, who pointed out that in considering the furanoseconformations which give rise to B-form duplexes consideration shouldalso be given to a 04′-endo pucker contribution.

DNA:RNA hybrid duplexes, however, are usually less stable than pureRNA:RNA duplexes, and depending on their sequence may be either more orless stable than DNA:DNA duplexes (Searle et al., Nucleic Acids Res.,1993, 21, 2051-2056). The structure of a hybrid duplex is intermediatebetween A- and B-form geometries, which may result in poor stackinginteractions (Lane et al., Eur. J. Biochem., 1993, 215, 297-306;Fedoroff et al., J. Mol. Biol., 1993, 233, 509-523; Gonzalez et al.,Biochemistry, 1995, 34, 4969-4982; Horton et al., J Mol. Biol., 1996,264, 521-533). The stability of the duplex formed between a target RNAand a synthetic sequence is central to therapies such as but not limitedto antisense and RNA interference as these mechanisms require thebinding of a synthetic oligomer strand to an RNA target strand. In thecase of antisense, effective inhibition of the mRNA requires that theantisense DNA have a very high binding affinity with the mRNA. Otherwisethe desired interaction between the synthetic oligomer strand and targetmRNA strand will occur infrequently, resulting in decreased efficacy.

One routinely used method of modifying the sugar puckering is thesubstitution of the sugar at the 2′-position with a substituent groupthat influences the sugar geometry. The influence on ring conformationis dependant on the nature of the substituent at the 2′-position. Anumber of different substituents have been studied to determine theirsugar puckering effect. For example, 2′-halogens have been studiedshowing that the 2′-fluoro derivative exhibits the largest population(65%) of the C3′-endo form, and the 2′-iodo exhibits the lowestpopulation (7%). The populations of adenosine (2′-OH) versusdeoxyadenosine (2′-H) are 36% and 19%, respectively. Furthermore, theeffect of the 2′-fluoro group of adenosine dimers(2′-deoxy-2′-fluoroadenosine-2′-deoxy-2′-fluoro-adenosine) is furthercorrelated to the stabilization of the stacked conformation.

As expected, the relative duplex stability can be enhanced byreplacement of 2′-OH groups with 2′-F groups thereby increasing theC3′-endo population. It is assumed that the highly polar nature of the2′-F bond and the extreme preference for C3′-endo puckering maystabilize the stacked conformation in an A-form duplex. Data from UVhypochromicity, circular dichroism, and ¹H NMR also indicate that thedegree of stacking decreases as the electronegativity of the halosubstituent decreases. Furthermore, steric bulk at the 2′-position ofthe sugar moiety is better accommodated in an A-form duplex than aB-form duplex. Thus, a 2′-substituent on the 3′-terminus of adinucleoside monophosphate is thought to exert a number of effects onthe stacking conformation: steric repulsion, furanose puckeringpreference, electrostatic repulsion, hydrophobic attraction, andhydrogen bonding capabilities. These substituent effects are thought tobe determined by the molecular size, electronegativity, andhydrophobicity of the substituent. Melting temperatures of complementarystrands is also increased with the 2′-substituted adenosinediphosphates. It is not clear whether the 3′-endo preference of theconformation or the presence of the substituent is responsible for theincreased binding. However, greater overlap of adjacent bases (stacking)can be achieved with the 3′-endo conformation.

One synthetic 2′-modification that imparts increased nuclease resistanceand a very high binding affinity to nucleotides is the 2-methoxyethoxy(2′-MOE, 2′-OCH₂CH₂OCH₃) side chain (Baker et al., J. Biol. Chem., 1997,272, 11944-12000). One of the immediate advantages of the 2′-MOEsubstitution is the improvement in binding affinity, which is greaterthan many similar 2′ modifications such as O-methyl, O-propyl, andO-aminopropyl. Oligonucleotides having the 2′-O-methoxyethyl substituentalso have been shown to be antisense inhibitors of gene expression withpromising features for in vivo use (Martin, P., Helv. Chim. Acta, 1995,78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al.,Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., NucleosidesNucleotides, 1997, 16, 917-926). Relative to DNA, the oligomers havingthe 2′-MOE modification displayed improved RNA affinity and highernuclease resistance. Chimeric oligomers having 2′-MOE substituents inthe wing nucleosides and an internal region of deoxy-phosphorothioatenucleotides (also termed a gapped oligomer or gapmer) have showneffective reduction in the growth of tumors in animal models at lowdoses. 2′-MOE substituted oligomers have also shown outstanding promiseas antisense agents in several disease states. One such MOE substitutedoligomer is presently being investigated in clinical trials for thetreatment of CMV retinitis.

Chemistries Defined

Unless otherwise defined herein, alkyl means C₁-C₁₂, preferably C₁-C₈,and more preferably C₁-C₆, straight or (where possible) branched chainaliphatic hydrocarbyl.

Unless otherwise defined herein, heteroalkyl means C₁-C₁₂, preferablyC₁-C₈, and more preferably C₁-C₆, straight or (where possible) branchedchain aliphatic hydrocarbyl containing at least one, and preferablyabout 1 to about 3, hetero atoms in the chain, including the terminalportion of the chain. Preferred heteroatoms include N, O and S.

Unless otherwise defined herein, cycloalkyl means C₃-C₁₂, preferablyC₃-C₈, and more preferably C₃-C₆, aliphatic hydrocarbyl ring.

Unless otherwise defined herein, alkenyl means C₂-C₁₂, preferably C₂-C₈,and more preferably C₂-C₆ alkenyl, which may be straight or (wherepossible) branched hydrocarbyl moiety, which contains at least onecarbon-carbon double bond.

Unless otherwise defined herein, alkynyl means C₂-C₁₂, preferably C₂-C₈,and more preferably C₂-C₆ alkynyl, which may be straight or (wherepossible) branched hydrocarbyl moiety, which contains at least onecarbon-carbon triple bond.

Unless otherwise defined herein, heterocycloalkyl means a ring moietycontaining at least three ring members, at least one of which is carbon,and of which 1, 2 or three ring members are other than carbon.Preferably the number of carbon atoms varies from 1 to about 12,preferably 1 to about 6, and the total number of ring members variesfrom three to about 15, preferably from about 3 to about 8. Preferredring heteroatoms are N, O and S. Preferred heterocycloalkyl groupsinclude morpholino, thiomorpholino, piperidinyl, piperazinyl,homopiperidinyl, homopiperazinyl, homomorpholino, homothiomorpholino,pyrrolodinyl, tetrahydrooxazolyl, tetrahydroimidazolyl,tetrahydrothiazolyl, tetrahydroisoxazolyl, tetrahydropyrrazolyl,furanyl, pyranyl, and tetrahydroisothiazolyl.

Unless otherwise defined herein, aryl means any hydrocarbon ringstructure containing at least one aryl ring. Preferred aryl rings haveabout 6 to about 20 ring carbons. Especially preferred aryl ringsinclude phenyl, napthyl, anthracenyl, and phenanthrenyl.

Unless otherwise defined herein, hetaryl means a ring moiety containingat least one fully unsaturated ring, the ring consisting of carbon andnon-carbon atoms. Preferably the ring system contains about 1 to about 4rings. Preferably the number of carbon atoms varies from 1 to about 12,preferably 1 to about 6, and the total number of ring members variesfrom three to about 15, preferably from about 3 to about 8. Preferredring heteroatoms are N, O and S. Preferred hetaryl moieties includepyrazolyl, thiophenyl, pyridyl, imidazolyl, tetrazolyl, pyridyl,pyrimidinyl, purinyl, quinazolinyl, quinoxalinyl, benzimidazolyl,benzothiophenyl, etc.

The term haloalkyl is defined as an alkyl containing one or more halogenatoms. In some embodiments, the alkyl is fully halogenated. For example,the haloalkyl may be trifluoromethyl. Similarly, the term haloalkoxy isdefined as an alkoxy group where the alkyl group is a haloalkyl. Forexample, the haloalkoxy may be trifluoroalkoxy.

Unless otherwise defined herein, where a moiety is defined as a compoundmoiety, such as hetarylalkyl (hetaryl and alkyl), aralkyl (aryl andalkyl), etc., each of the sub-moieties is as defined herein.

Unless otherwise defined herein, an electron withdrawing group is agroup, such as the cyano or isocyanato group that draws electroniccharge away from the carbon to which it is attached.

Other electron withdrawing groups of note include those whoseelectronegativities exceed that of carbon, for example halogen, nitro,or phenyl substituted in the ortho- or para-position with one or morecyano, isothiocyanato, nitro or halo groups.

Unless otherwise defined herein, the terms halogen and halo have theirordinary meanings. Preferred halo (halogen) substituents are Cl, Br, andI.

The aforementioned optional substituents are, unless otherwise hereindefined, suitable substituents depending upon desired properties.Included are halogens (Cl, Br, I), alkyl, alkenyl, and alkynyl moieties,NO₂, NH₃ (substituted and unsubstituted), acid moieties (e.g. —CO₂H,—OSO₃H₂, etc.), heterocycloalkyl moieties, hetaryl moieties, arylmoieties, etc.In all the preceding formulae, the squiggle (˜) indicates a bond to anoxygen or sulfur of the 5′-phosphate.

Phosphate protecting groups include those described in U.S. Pat. Nos.5,760,209, 5,614,621, 6,051,699, 6,020,475, 6,326,478, 6,169,177,6,121,437, 6,465,628 each of which is expressly incorporated herein byreference in its entirety.

Phosphotioate groups include those described in U.S. Pat. Nos.3,687,808, 5,188,897, 5,278,302, 5,286,717, 5,405,939, 5,453,496, and5,587,361.

Alkylphosphoroamidate groups include those described in U.S. Pat. Nos.5,536,821 and 5,541,306,

Unless otherwise defined herein, alkoxy is defined as —O-alkyl wherealkyl is as defined above.

Unless otherwise defined herein, alkylthio is defined as —S-alkyl wherealkyl is as defined above.

As used herein, the terms alkyl, heteroalkyl, cycloalkyl, alkenyl,alkynyl, heterocycloalkyl, aryl, and hetaryl include moieties that areoptionally substituted. Sitable substituents are well known to thoseskilled in the art. These substituents include C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, C₅-C₂₀ aryl, —O-alkyl, —O-alkenyl, —O-alkynyl,—O-alkylamino, —O-alkylalkoxy, —O— alkylaminoalkyl, —O-alkyl imidazole,—OH, —SH, —S-alkyl, —S-alkenyl, —S-alkynyl, —N(H)-alkyl, —N(H)-alkenyl,—N(H)-alkynyl, —N(alkyl)₂, —O-aryl, —S-aryl, —NH-aryl, —ONO₂,—O-aralkyl, —S-aralkyl, —N(H)-aralkyl, phthalimido (attached at N),halogen, amino, keto (—C(═O)—R), carboxyl (—C(═O)OH), nitro (—NO₂),nitroso (—N═O), cyano (—CN), trifluoromethyl (—CF₃), trifluoromethoxy(—O—CF₃), imidazole, azido (—N₃), hydrazino (—N(H)—NH₂), aminooxy(—O—NH₂), isocyanato (—N═C═O), sulfoxide (—S(═O)—R), sulfone(—S(═O)₂—R), disulfide (—S—S—R), silyl, heterocycle, carbocycle,intercalator, reporter group, conjugate, polyamine, polyamide,polyalkylene glycol, and polyethers of the formula (—O-alkyl)_(m), wherem is 1 to about 10; wherein each R is, independently, hydrogen, aprotecting group alkyl, alkenyl, or alkynyl.

The terms blockmer, 3′-hemimer, 5′-hemimer, gapmer and inverted gapmerare used in this specification to identify certain motifs or positionalplacement of types or segments of nucleotides in an oligomer. Dependingon the number of nucleotide or nucleoside subunits and their position inthe oligomer, one or more than one of these terms might apply to aparticular construction and could be used for identification purposes. Ablockmer has at least one block or segment of at least two consecutivelylocated nucleotide or nucleoside subunits of a first type positionedadjacent to at least one nucleotide or nucleoside of a second type. Thusfor instances if the nucleotides or nucleosides of the first type arerepresented by “X” and those of the second type are represented by“Y“and if” . . . ” represent nucleotides or nucleosides other that the Xor Y type nucleotides or the absence of any nucleotides then thefollowing structures . . . XXY . . . ; . . . XXYXX . . . ; . . . XXYXXY. . . ; . . . XXYXXYXX . . . on so on for higher homologs are possiblewhere each X containing segment includes two members and each Ycontaining segment includes only one member. If each X containingsegment includes two members and each Y subunit also includes twomembers other representational blockmers include . . . XXYY . . . ; . .. XXYYXX . . . ; . . . XXYYXXYY . . . ; . . . XXYYXXYYXX . . . and so onfor high homologs. These can be extended to other representativestructures having more X and/or Y members in the blocks or segments, asfor instances the structures YXXXXYYYXXXXY; YYXXXYYXXXXYY; andYYYYXXXXYYYYXXXX.

If a block or segment of the first type of nucleotides or nucleosideresides at the 5′ or the 3′ terminus and all of the remainingnucleotides or nucleosides of the oligomer are of the second type, thenthat blockmer is also a hemimer. Using the same X and Y representationand selecting five members for each segment and basing the hemimerdesignation on the X members then the representations XXXXXYYYYY andYYYYYXXXX represent, respectively, 5′ and 3′ hemimers.

In gapmers, a block or segment of one type of nucleotides or nucleosidesis interspaced between first and second blocks of the second type. Asbefore if the designation is based on the X members then XXXXYYYYXXXXrepresents a gapmer and YYYYXXXXYYYY represents an invertered gapmer.

Screening, Target Validation and Drug Discovery

For use in screening and target validation, the compounds andcompositions of the invention are used to modulate the expression of aselected protein. “Modulators” are those oligomeric compounds andcompositions that decrease or increase the expression of a nucleic acidmolecule encoding a protein and which comprise at least an 8-nucleobaseportion which is complementary to a preferred target segment. Thescreening method comprises the steps of contacting a preferred targetsegment of a nucleic acid molecule encoding a protein with one or morecandidate modulators, and selecting for one or more candidate modulatorswhich decrease or increase the expression of a nucleic acid moleculeencoding a protein. Once it is shown that the candidate modulator ormodulators are capable of modulating (e.g. either decreasing orincreasing) the expression of a nucleic acid molecule encoding apeptide, the modulator may then be employed in further investigativestudies of the function of the peptide, or for use as a research,diagnostic, or therapeutic agent in accordance with the presentinvention.

The conduction such screening and target validation studies, oligomericcompounds of invention can be used combined with their respectivecomplementary strand oligomeric compound to form stabilizeddouble-stranded (duplexed) oligomers. Double stranded oligomer moietieshave been shown to modulate target expression and regulate translationas well as RNA processing via an antisense mechanism. Moreover, thedouble-stranded moieties may be subject to chemical modifications (Fireet al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395,854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science,1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998,95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197;Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev.2001, 15, 188-200; Nishikura et al., Cell (2001), 107, 415-416; and Basset al., Cell (2000), 101, 235-238.) For example, such double-strandedmoieties have been shown to inhibit the target by the classicalhybridization of antisense strand of the duplex to the target, therebytriggering enzymatic degradation of the target (Tijsterman et al.,Science, 2002, 295, 694-697).

For use in drug discovery and target validation, oligomeric compounds ofthe present invention are used to elucidate relationships that existbetween proteins and a disease state, phenotype, or condition. Thesemethods include detecting or modulating a target peptide comprisingcontacting a sample, tissue, cell, or organism with the oligomericcompounds and compositions of the present invention, measuring thenucleic acid or protein level of the target and/or a related phenotypicor chemical endpoint at some time after treatment, and optionallycomparing the measured value to a non-treated sample or sample treatedwith a further oligomeric compound of the invention. These methods canalso be performed in parallel or in combination with other experimentsto determine the function of unknown genes for the process of targetvalidation or to determine the validity of a particular gene product asa target for treatment or prevention of a disease or disorder.

Kits, Research Reagents, Diagnostics, and Therapeutics

The oligomeric compounds and compositions of the present invention canadditionally be utilized for diagnostics, therapeutics, prophylaxis andas research reagents and kits. Such uses allows for those of ordinaryskill to elucidate the function of particular genes or to distinguishbetween functions of various members of a biological pathway.

For use in kits and diagnostics, the oligomeric compounds andcompositions of the present invention, either alone or in combinationwith other compounds or therapeutics, can be used as tools indifferential and/or combinatorial analyses to elucidate expressionpatterns of a portion or the entire complement of genes expressed withincells and tissues.

As one non-limiting example, expression patterns within cells or tissuestreated with one or more compounds or compositions of the invention arecompared to control cells or tissues not treated with the compounds orcompositions and the patterns produced are analyzed for differentiallevels of gene expression as they pertain, for example, to diseaseassociation, signaling pathway, cellular localization, expression level,size, structure or function of the genes examined. These analyses can beperformed on stimulated or unstimulated cells and in the presence orabsence of other compounds that affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480,17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serialanalysis of gene expression)(Madden, et al., Drug Discov. Today, 2000,5, 415-425), READS (restriction enzyme amplification of digested cDNAs)(Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (totalgene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci.U.S.A, 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999,20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al.,FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80,143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,203-208), subtractive cloning, differential display (DD) (Jurecic andBelmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomichybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31,286-96), FISH (fluorescent in situ hybridization) techniques (Going andGusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometrymethods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

The compounds and compositions of the invention are useful for researchand diagnostics, because these compounds and compositions hybridize tonucleic acids encoding proteins. Hybridization of the compounds andcompositions of the invention with a nucleic acid can be detected bymeans known in the art. Such means may include conjugation of an enzymeto the compound or composition, radiolabelling or any other suitabledetection means. Kits using such detection means for detecting the levelof selected proteins in a sample may also be prepared.

The specificity and sensitivity of compounds and compositions can alsobe harnessed by those of skill in the art for therapeutic uses.Antisense oligomeric compounds have been employed as therapeuticmoieties in the treatment of disease states in animals, includinghumans. Antisense oligomer drugs, including ribozymes, have been safelyand effectively administered to humans and numerous clinical trials arepresently underway. It is thus established that oligomeric compounds canbe useful therapeutic modalities that can be configured to be useful intreatment regimes for the treatment of cells, tissues and animals,especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder that can be treated by modulating the expression ofa selected protein is treated by administering the compounds andcompositions. For example, in one non-limiting embodiment, the methodscomprise the step of administering to the animal in need of treatment, atherapeutically effective amount of a protein inhibitor. The proteininhibitors of the present invention effectively inhibit the activity ofthe protein or inhibit the expression of the protein. In one embodiment,the activity or expression of a protein in an animal is inhibited byabout 10%. Preferably, the activity or expression of a protein in ananimal is inhibited by about 30%. More preferably, the activity orexpression of a protein in an animal is inhibited by 50% or more.

For example, the reduction of the expression of a protein may bemeasured in serum, adipose tissue, liver or any other body fluid, tissueor organ of the animal. Preferably, the cells contained within thefluids, tissues or organs being analyzed contain a nucleic acid moleculeencoding a protein and/or the protein itself.

The compounds and compositions of the invention can be utilized inpharmaceutical compositions by adding an effective amount of thecompound or composition to a suitable pharmaceutically acceptablediluent or carrier. Use of the oligomeric compounds and methods of theinvention may also be useful prophylactically.

Formulations

The compounds and compositions of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S.: 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The compounds and compositions of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal, including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to prodrugs and pharmaceutically acceptablesalts of the oligomeric compounds of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligomers of the invention are prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives according to the methods disclosed in WO 93/24510to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S.Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds and compositionsof the invention: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto. For oligomers, preferred examples ofpharmaceutically acceptable salts and their uses are further describedin U.S. Pat. No. 6,287,860, which is incorporated herein in itsentirety.

The present invention also includes pharmaceutical compositions andformulations that include the compounds and compositions of theinvention. The pharmaceutical compositions of the present invention maybe administered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compounds and compositions of the present invention may beformulated into any of many possible dosage forms such as, but notlimited to, tablets, capsules, gel capsules, liquid syrups, soft gels,suppositories, and enemas. The compositions of the present invention mayalso be formulated as suspensions in aqueous, non-aqueous or mixedmedia. Aqueous suspensions may further contain substances which increasethe viscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug that may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligomers. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligomers of the invention are in admixture with a topical deliveryagent such as lipids, liposomes, fatty acids, fatty acid esters,steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, compounds and compositions of theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, they may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Topical formulations are describedin detail in U.S. patent application Ser. No. 09/315,298 filed on May20, 1999, which is incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligomers of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Compounds and compositions of theinvention may be delivered orally, in granular form including sprayeddried particles, or complexed to form micro or nanoparticles. Complexingagents and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety. Certain oral formulationsfor oligomers and their preparation are described in detail in U.S.application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No.09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8,2002, each of which is incorporated herein by reference in theirentirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more of the compounds and compositions of theinvention and one or more other chemotherapeutic agents that function bya non-antisense mechanism. Examples of such chemo-therapeutic agentsinclude but are not limited to cancer chemotherapeutic drugs such asdaunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosinearabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C,actinomycin D, mithramycin, prednisone, hydroxyprogesterone,testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the oligomeric compounds of the invention, suchchemo-therapeutic agents may be used individually (e.g., 5-FU andoligomer), sequentially (e.g., 5-FU and oligomer for a period of timefollowed by MTX and oligomer), or in combination with one or more othersuch chemotherapeutic agents (e.g., 5-FU, MTX and oligomer, or 5-FU,radiotherapy and oligomer). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of theinvention. Combinations of compounds and compositions of the inventionand other drugs are also within the scope of this invention. Two or morecombined compounds such as two oligomeric compounds or one oligomericcompound combined with further compounds may be used together orsequentially.

In another related embodiment, compositions of the invention may containone or more of the compounds and compositions of the invention targetedto a first nucleic acid and one or more additional compounds such asantisense oligomeric compounds targeted to a second nucleic acid target.Numerous examples of antisense oligomeric compounds are known in theart. Alternatively, compositions of the invention may contain two ormore oligomeric compounds and compositions targeted to different regionsof the same nucleic acid target. Two or more combined compounds may beused together or sequentially

Dosing

The formulation of therapeutic compounds and compositions of theinvention and their subsequent administration (dosing) is believed to bewithin the skill of those in the art. Dosing is dependent on severityand responsiveness of the disease state to be treated, with the courseof treatment lasting from several days to several months, or until acure is effected or a diminution of the disease state is achieved.Optimal dosing schedules can be calculated from measurements of drugaccumulation in the body of the patient. Persons of ordinary skill caneasily determine optimum dosages, dosing methodologies and repetitionrates. Optimum dosages may vary depending on the relative potency ofindividual oligomers, and can generally be estimated based on EC₅₀sfound to be effective in in vitro and in vivo animal models. In general,dosage is from 0.01 ug to 100 g per kg of body weight, and may be givenonce or more daily, weekly, monthly or yearly, or even once every 2 to20 years. Persons of ordinary skill in the art can easily estimaterepetition rates for dosing based on measured residence times andconcentrations of the drug in bodily fluids or tissues. Followingsuccessful treatment, it may be desirable to have the patient undergomaintenance therapy to prevent the recurrence of the disease state,wherein the oligomer is administered in maintenance doses, ranging from0.01 ug to 100 g per kg of body weight, once or more daily, to onceevery 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

The entire disclosure of each patent, patent application, andpublication cited or described in this document is hereby incorporatedby reference.

Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite,5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites,5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O—[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine and5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated byreference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference. 3′-Deoxy-3′-amino phosphoramidate oligonucleotides areprepared as described in U.S. Pat. No. 5,476,925, herein incorporated byreference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P═O or P═S linkages are prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289,all of which are herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S2Na2)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe,S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M.D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191;Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22,1859-1862; Dahl, B. J., et al., Acta Chem. Scand. 1990, 44, 639-641;Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott,F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., etal., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al.,Tetrahedron, 1967, 23, 2315-2331).

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-O-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Synthesis of 2′-Deoxy-2′-fluoro Modified Oligonucleotides

2′-Deoxy-2′-fluoro modified oligonucleotides may be prepared by methodstaught in U.S. Pat. No. 6,531,584.

Example 6 Synthesis of 2′-Deoxy-2′-O-alkyl Modified Oligonucleotides

2′-Deoxy-2′-O-alkyl modified oligonucleotides may be prepared by methodstaught in U.S. Pat. No. 6,531,584.

Example 7 Synthesis of 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methylUridine

2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine may beprepared by methods taught in U.S. Pat. No. 6,043,352.

Example 8 Synthesis of5′-O-Dimethoxytrityl-2′-O-methyl-3′-O—(N,N-diisopropylamino-O-β-cyanoethylphosphine)-N-benzoyladenosine

5′-O-Dimethoxytrityl-2′-O-methyl-3′-O—(N,N-diisopropylamino-O-.beta.-cyanoethylphosphine)-N-benzoyladenosinemay be prepared by methods taught inU.S. Pat. No. 6,005,094.

Example 9 Synthesis of5′-O-Dimethoxytrityl-2′-O-Methylthiomethyl-Nucleotides

5′-O-Dimethoxytrityl-2′-O-methylthiomethyl-nucleotides may be preparedby methods taught in U.S. Pat. No. 6,239,272.

Example 10 Synthesis of 2′-Deoxy-2′-(vinyloxy) Modified Oligonucleotides

2′-Deoxy-2′-(vinyloxy) modified oligonucleotides may be prepared bymethods taught in U.S. Pat. No. 5,859,221.

Example 11 Synthesis of 2′-Deoxy-2′-(methylthio), (methylsulfinyl) and(methylsulfonyl) Modified Oligonucleotides

2′-Deoxy-2′-(methylthio), (methylsulfinyl) and (methylsulfonyl) modifiedoligonucleotides may be prepared by methods taught in U.S. Pat. No.5,859,221.

Example 12 Synthesis of Oligonucleotides Bearing 2′-OCH₂COOEtSubstituents

2′-OCH₂COOEt modified oligonucleotides may be prepared by methods taughtin U.S. Pat. No. 5,792,847.

Example 13 Synthesis of 9-(2-(O-2-Propynyloxy)-β-D-ribofuranosyl)Adenine

9-(2-(O-2-Propynyloxy)-β-D-ribofuranosyl) adenine may be prepared bymethods taught in U.S. Pat. No. 5,514,786.

Example 14 Synthesis of3′-O—(N-Allyloxycarbonyl-6-aminohexyl)-5′-O-dimethoxytrityl-uridine

3′-O—(N-Allyloxycarbonyl-6-aminohexyl)-5′-O-dimethoxytrityl-uridine maybe prepared by methods taught in U.S. Pat. No. 6,111,085.

Example 15 Synthesis of 2′-O—(N-phthalimido) prop-3-yl adenosine

2′-O—(N-phthalimido) prop-3-yl adenosine may be prepared by methodstaught in U.S. Pat. No. 5,872,232.

Example 16 Synthesis of2′-O-(2-Phthalimido-N-hydroxyethyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosine

2′-O-(2-Phthalimido-N-hydroxyethyl)-3′,5′-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)adenosinemay be prepared by methods taught in U.S. Pat. No. 6,172,209.

Example 17 Synthesis of 5′-O-Dimethoxytrityl-2′-O-(carbonylaminohexylaminocarbonyloxy cholesteryl)-N₄-benzolyl chloride

5′-O-Dimethoxytrityl-2′-O-(carbonylaminohexyl aminocarbonyloxycholesteryl)-N4-benzolyl chloride may be prepared by methods taught inU.S. Pat. No. 6,166,188.

Example 18 Synthesis of5′-O-[(2,2-dimethyl-1,1-diphenyl-1-silapropoxy)methyl]-2′-O—((N,N-dimethylaminoethyleneamino)carbonylmethylene)adenosine

5′-O-[(2,2-dimethyl-1,1-diphenyl-1-silapropoxy)methyl]-2′-O—((N,N-dimethylaminoethyleneamino)carbonylmethylene)adenosinemay be prepared by methods taught in U.S. Pat. No. 6,147,200.

Example 19 Synthesis of 2′-O-(Propylsulfonic acid) SodiumSalt-N-3-(Benzyloxy) Methyl-5-Methyluridine

2′-O-(Propylsulfonic acid) sodium salt-N-3-(benzyloxy)methyl-5-methyluridine may be prepared by methods taught in U.S. Pat.No. 6,277,982.

Example 20 Synthesis of

Modified Oligonucleotides

These oligonucleotides may be prepared by methods taught in U.S. Pat.No. 5,969,116.

Example 21 Synthesis of5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyl Uridine

5′-Dimethoxytrityl-2′-O-(trans-2-methoxycyclohexyl)-5-methyl uridine maybe prepared by methods taught in U.S. Pat. No. 6,277,982.

Example 22 Synthesis of 2′-OH, 2′-Me Modified Compounds

The above compound was prepared following the methods described in J.Med. Chem. 41: 1708 (1998).

Example 23 4-Amino-7-(2-C-methyl-β-D-arabinofuranosyl)-7H-pyrrolo[2,3-d]pyrimidine

To CrO₃ (1.57 g, 1.57 mmol) in dichloromethane (DCM) (10 mL) at 0° C.was added acetic anhydride (145 mg, 1.41 mmol) and then pyridine (245mg, 3.10 mmol). The mixture was stirred for 15 min, then a solution of7-[3,5-O-[1,1,3,3-tetrakis(1-methylethyl)-1,3-disiloxanediyl]-β-D-ribofuranosyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine[for preparation, see J. Am. Chem. Soc. 105: 4059 (1983)] (508 mg, 1.00mmol) in DCM (3 mL) was added. The resulting solution was stirred for 2h and then poured into ethyl acetate (10 mL), and subsequently filteredthrough silica gel using ethyl acetate as the eluent. The combinedfiltrates were evaporated in vacuo, taken up in diethyl ether/THF (1:1)(20 mL), cooled to −78° C. and methylmagnesium bromide (3M, in THF)(3.30 mL, 10 mmol) was added dropwise. The mixture was stirred at −78°C. for 10 min, then allowed to come to room temperature (rt) andquenched by addition of saturated aqueous ammonium chloride (10 mL) andextracted with DCM (20 mL). The organic phase was evaporated in vacuoand the crude product purified on silica gel using 5% methanol indichloromethane as eluent. Fractions containing the product were pooledand evaporated in vacuo. The resulting oil was taken up in THF (5 mL)and tetrabutylammonium fluoride (TBAF) on silica (1.1 mmol/g on silica)(156 mg) was added. The mixture was stirred at rt for 30 min, filtered,and evaporated in vacuo. The crude product was purified on silica gelusing 10% methanol in dichloromethane as eluent. Fractions containingthe product were pooled and evaporated in vacuo to give the desiredcompound (49 mg) as a colorless solid.

¹H NMR (DMSO-d₆): δ 1.08 (s, 3H), 3.67 (m, 2H), 3.74 (m, 1H), 3.83 (m,1H), 5.19 (m, 1H), 5.23 (m, 1H), 5.48 (m, 1H), 6.08 (1H, s), 6.50 (m,1H), 6.93 (bs, 2H), 7.33 (m, 1H), 8.02 (s, 1H).

Example 24 Design and Screening of Duplexed Oligomeric CompoundsTargeting a Target

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense oligomeric compounds of the presentinvention and their complements can be designed to target a target. Theends of the strands may be modified by the addition of one or morenatural or modified nucleobases to form an overhang. The sense strand ofthe dsRNA is then designed and synthesized as the complement of theantisense strand and may also contain modifications or additions toeither terminus. For example, in one embodiment, both strands of thedsRNA duplex would be complementary over the central nucleobases, eachhaving overhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG (SEQ ID NO: 1) and having a two-nucleobase overhangof deoxythymidine(dT) would have the following structure:

5′     c g a g a g g c g g a c g g g a c c g T T 3′ Antisense Strand(SEQ ID NO: 2)        | | | | | | | | | | | | | | | | | | | 3′T T g c t c t c c g c c t g c c c t g g c     5′ Complément Strand(SEQ ID NO: 3)

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense oligomeric compounds are evaluatedfor their ability to modulate a target expression.

When cells reached 80% confluency, they are treated with duplexedantisense oligomeric compounds of the invention. For cells grown in96-well plates, wells are washed once with 200 μL OPTI-MEM-1reduced-serum medium (Gibco BRL) and then treated with 130 μL ofOPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desiredduplex antisense oligomeric compound at a final concentration of 200 nM.After 5 hours of treatment, the medium is replaced with fresh medium.Cells are harvested 16 hours after treatment, at which time RNA isisolated and target reduction measured by RT-PCR.

Example 25 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32+/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 1991, 266, 18162-18171. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 26 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 27 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition wasconfirmed by mass analysis of the oligomeric compounds utilizingelectrospray-mass spectroscopy. All assay test plates were diluted fromthe master plate using single and multi-channel robotic pipettors.Plates were judged to be acceptable if at least 85% of the oligomericcompounds on the plate were at least 85% full length.

Example 28 Cell Culture and Oligonucleotide Treatment

The effect of oligomeric compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin100 units per mL, and streptomycin 100 micrograms per mL (InvitrogenCorporation, Carlsbad, Calif.). Cells were routinely passaged bytrypsinization and dilution when they reached 90% confluence. Cells wereseeded into 96-well plates (Falcon-Primaria #353872) at a density of7000 cells/well for use in RT-PCR analysis.

For Northern blotting or other analysis, cells may be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with Antisense Oligomeric Compounds:

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM™-1 reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL ofOPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation,Carlsbad, Calif.) and the desired concentration of oligonucleotide.Cells are treated and data are obtained in triplicate. After 4-7 hoursof treatment at 37° C., the medium was replaced with fresh medium. Cellswere harvested 16-24 hours after oligonucleotide treatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 4) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 5) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 6, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 29 Analysis of Oligonucleotide Inhibition of a Target Expression

Modulation of a target expression can be assayed in a variety of waysknown in the art. For example, a target mRNA levels can be quantitatedby, e.g., Northern blot analysis, competitive polymerase chain reaction(PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR ispresently preferred. RNA analysis can be performed on total cellular RNAor poly(A)+mRNA. The preferred method of RNA analysis of the presentinvention is the use of total cellular RNA as described in otherexamples herein. Methods of RNA isolation are well known in the art.Northern blot analysis is also routine in the art. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM™ 7600, 7700, or 7900 Sequence DetectionSystem, available from PE-Applied Biosystems, Foster City, Calif. andused according to manufacturer's instructions.

Protein levels of a target can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

Example 30 Design of Phenotypic Assays and In Vivo Studies for the Useof a Target Inhibitors Phenotypic Assays

Once a target inhibitors have been identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with atarget inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the geneotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the target inhibitors. Hallmarkgenes, or those genes suspected to be associated with a specific diseasestate, condition, or phenotype, are measured in both treated anduntreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study.

To account for the psychological effects of receiving treatments,volunteers are randomly given placebo or a target inhibitor.Furthermore, to prevent the doctors from being biased in treatments,they are not informed as to whether the medication they areadministering is a a target inhibitor or a placebo. Using thisrandomization approach, each volunteer has the same chance of beinggiven either the new treatment or the placebo.

Volunteers receive either the a target inhibitor or placebo for eightweek period with biological parameters associated with the indicateddisease state or condition being measured at the beginning (baselinemeasurements before any treatment), end (after the final treatment), andat regular intervals during the study period. Such measurements includethe levels of nucleic acid molecules encoding a target or a targetprotein levels in body fluids, tissues or organs compared topre-treatment levels. Other measurements include, but are not limitedto, indices of the disease state or condition being treated, bodyweight, blood pressure, serum titers of pharmacologic indicators ofdisease or toxicity as well as ADME (absorption, distribution,metabolism and excretion) measurements.

Information recorded for each patient includes age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and a target inhibitor treatment. In general,the volunteers treated with placebo have little or no response totreatment, whereas the volunteers treated with the target inhibitor showpositive trends in their disease state or condition index at theconclusion of the study.

Example 31 RNA Isolation Poly(A)+mRNA Isolation

Poly(A)+mRNA was isolated according to Miura et al., (Clin. Chem., 1996,42, 1758-1764). Other methods for poly(A)+mRNA isolation are routine inthe art. Briefly, for cells grown on 96-well plates, growth medium wasremoved from the cells and each well was washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, theplate was gently agitated and then incubated at room temperature forfive minutes. 55 μL of lysate was transferred to Oligo d(T) coated96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60minutes at room temperature, washed 3 times with 200 μL of wash buffer(10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash,the plate was blotted on paper towels to remove excess wash buffer andthen air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH7.6), preheated to 70° C., was added to each well, the plate wasincubated on a 90° C. hot plate for 5 minutes, and the eluate was thentransferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96™ kit and buffers purchasedfrom Qiagen Inc. (Valencia, Calif.) following the manufacturer'srecommended procedures. Briefly, for cells grown on 96-well plates,growth medium was removed from the cells and each well was washed with200 μL cold PBS. 150 μL Buffer RLT was added to each well and the platevigorously agitated for 20 seconds. 150 μL of 70% ethanol was then addedto each well and the contents mixed by pipetting three times up anddown. The samples were then transferred to the RNEASY 96™ well plateattached to a QIAVAC™ manifold fitted with a waste collection tray andattached to a vacuum source. Vacuum was applied for 1 minute. 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and incubatedfor 15 minutes and the vacuum was again applied for 1 minute. Anadditional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE wasthen added to each well of the RNEASY 96™ plate and the vacuum appliedfor a period of 90 seconds. The Buffer RPE wash was then repeated andthe vacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC™ manifold and blotted dry on paper towels. Theplate was then re-attached to the QIAVAC™ manifold fitted with acollection tube rack containing 1.2 mL collection tubes. RNA was theneluted by pipetting 140 μL of RNAse free water into each well,incubating 1 minute, and then applying the vacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 32

Real-Time Quantitative PCR Analysis of a Target mRNA Levels

Quantitation of a target mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 UnitsMuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction wascarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol were carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RiboGreen™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Probes and primers are designed to hybridize to a human a targetsequence, using published sequence information.

Example 33

Northern Blot Analysis of a Target mRNA Levels

Eighteen hours after treatment, cell monolayers were washed twice withcold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood,Tex.). Total RNA was prepared following manufacturer's recommendedprotocols. Twenty micrograms of total RNA was fractionated byelectrophoresis through 1.2% agarose gels containing 1.1% formaldehydeusing a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA wastransferred from the gel to HYBOND™-N+ nylon membranes (AmershamPharmacia Biotech, Piscataway, N.J.) by overnight capillary transferusing a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc.,Friendswood, Tex.). RNA transfer was confirmed by UV visualization.Membranes were fixed by UV cross-linking using a STRATALINKER™ UVCrosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probedusing QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.)using manufacturer's recommendations for stringent conditions.

To detect human a target, a human a target specific primer probe set isprepared by PCR To normalize for variations in loading and transferefficiency membranes are stripped and probed for humanglyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, PaloAlto, Calif.).

Hybridized membranes were visualized and quantitated using aPHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics,Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreatedcontrols.

Example 34 Inhibition of Human a Target Expression by Oligonucleotides

In accordance with the present invention, a series of oligomericcompounds are designed to target different regions of the human targetRNA. The oligomeric compounds are analyzed for their effect on humantarget mRNA levels by quantitative real-time PCR as described in otherexamples herein. Data are averages from three experiments. The targetregions to which these preferred sequences are complementary are hereinreferred to as “preferred target segments” and are therefore preferredfor targeting by oligomeric compounds of the present invention. Thesequences represent the reverse complement of the preferred antisenseoligomeric compounds.

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisenseoligomeric compounds of the present invention, one of skill in the artwill recognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother oligomeric compounds that specifically hybridize to thesepreferred target segments and consequently inhibit the expression of atarget.

According to the present invention, antisense oligomeric compoundsinclude antisense oligomeric compounds, antisense oligonucleotides,ribozymes, external guide sequence (EGS) oligonucleotides, alternatesplicers, primers, probes, and other short oligomeric compounds thathybridize to at least a portion of the target nucleic acid.

Example 35 Western Blot Analysis of a Target Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to a target is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 36

Blockmer Walk of 5 2′-O-Methy Modified Nucleosides in the AntisenseStrand of siRNA's Assayed for PTEN mRNA Levels Against Untreated Control

The antisense (AS) strands listed below having SEQ ID NO: 8 wereindividually duplexed with the sense (S) strand having SEQ ID NO: 7 andthe activity was measured to determine the relative positional effect ofthe 5 modifications.

SEQ ID NO:/ ISIS NO Sequence 7/271790(S) 5′-CAAAUCCAGAGGCUAGCAG-dTdT-3′8/271071(AS) 3′-dTdT-GUUUAGGUCUCCGAUCGUC-5′ 8/271072(AS)3′-dTdT-GUUUAGGUCUCCGAUCGUC-5′ 8/271073(AS)3′-dTdT-GUUUAGGUCUCCGAUCGUC-5′ 8/271074(AS)3′-dTdT-GUUUAGGUCUCCGAUCGUC-5′ 8/271075(AS)3′-dTdT-GUUUAGGUCUCCGAUCGUC-5′

Underlined nucleosides are 2′-O-methyl modified nucleosides, dT's aredeoxy thymidines, all other nucleosides are ribonucleosides and allinternucleoside linkages are phosphodiester.

The siRNA's having 5, 2′-O-methyl groups at least 2 positions removedfrom the 5′-end of the antisense strand reduced PTEN mRNA levels to from25 to 35% of untreated control. The remaining 2 constructs increasedPTEN mRNA levels above untreated control.

Example 37

Solid Block of 2′-O-Methyl Modified Nucleosides in the Antisense Strandof siRNA's Assayed for PTEN mRNA Levels Against Untreated Control

The antisense strands listed below having SEQ ID NO:9 were individuallyduplexed with the sense strand having SEQ ID NO:7 and the activity wasmeasured to determine the relative effect of adding either 9 or 14,2′-O-methyl modified nucleosides at the 3′-end of the resulting siRNA's.

SEQ ID NO:/ ISIS NO Sequence 7/271790(S) 5′-CAAAUCCAGAGGCUAGCAG-dTdT-3′9/271079(AS) 3′-UUGUUUAGGUCUCCGAUCGUC-5′ 9/271081(AS)3′-UUGUUUAGGUCUCCGAUCGUC-5′

Underlined nucleosides are 2′-O-methyl modified nucleosides, dT's aredeoxy thymidines, all other nucleosides are ribonucleosides and allinternucleoside linkages are phosphodiester.

SEQ ID NO: Sequence (5′-3′) 9 CUGCUAGCCUCUGGAUUUGUU

The siRNA having 9, 2′-O-methyl nucleosides reduced PTEN mRNA levels toabout 40% of untreated control whereas the construct having 14,2′-O-methyl nucleosides only reduced PTEN mRNA levels to about 98% ofcontrol.

Example 38

2′-O-Methy Blockmers (siRNA Vs asRNA)

A series of blockmers were prepared as duplexed siRNA's and also assingle strand asRNA's. The antisense strands were identical for thesiRNA's and the asRNA's.

SEQ ID NO:/ ISIS NO Sequence 5′-3′ 10/308746 (S)5′-AAGUAAGGACCAGAGACAAA-3′ (PO) 11/303912 (AS)3′-UUCAUUCCUGGUCUCUGUUU-P 5′ (PS) 11/316449 (AS)3′-UUCAUUCCUGGUCUCUGUUU-P 5′ (PS) 11/335223 (AS)3′-UUCAUUCCUGGUCUCUGUUU-P 5′ (PS) 11/335224 (AS)3′-UUCAUUCCUGGUCUCUGUUU-P 5′ (PS) 11/335225 (AS)3′-UUCAUUCCUGGUCUCUGUUU-P 5′ (PS) 11/335226 (AS)3′-UUCAUUCCUGGUCUCUGUUU-P 5′ (PS) 11/335227 (AS)3′-UUCAUUCCUGGUCUCUGUUU-P 5′ (PS) 11/335228 (AS)3′-UUCAUUCCUGGUCUCUGUUU-P 5′ (PS)

Underlined nucleosides are 2′-O-methyl modified nucleosides, all othernucleosides are ribonucleosides and all internucleoside linkages for theAS strands are phosphorothioate and the internucleoside linkages for theS strand are phosphodiester.

SEQ ID NO: Sequence (5′-3′) 10 AAGUAAGGACCAGAGACAAA 11UUUGUCUCUGGUCCUUACUU

The constructs were assayed for activity for measuring the levels ofPTEN mRNA in T24 cells against untreated control levels. All of theasRNA's and siRNA's showed activity with the asRNA's having the bestactivity in each case. A clear dose response was seen for all the siRNAconstructs (20, 40, 80 and 150 nm doses). There was a good dose responsefor the asRNA's for 50, 100 and 200 nm doses. In general the siRNA'swere more active in this system at lower doses than the asRNA's and atthe 150 nm dose was able to reduce PTEN mRNA levels to from 15 to 40% ofuntreated control. The unmodified siRNA 303912 reduced PTEN mRNA levelsto about 19% of the untreated control.

Example 39

3′-Hemimer 2′-O-Methyl siRNA Constructs

Blunt and overhanging siRNA constructs were prepared having a block of5, 2′-O-methyl nucleosides at the 3′-terminus.

SEQ ID NO:/ ISIS NO Sequence (overhangs)  7/271790(S)5′-CAAAUCCAGAGGCUAGCAG-dTdT-3′  9/xxxxxx (AS)3′-UUGUUUAGGUCUCCGAUCGUC-5′ Sequence (blunt) 12/xxxxx (S)5′-GUCAAAUCCAGAGGCUAGCAG-3′ 13/xxxxxx (AS) 3′-CAGUUUAGGUCUCCGAUCGUC-5′

Underlined nucleosides are 2′-O-methyl modified nucleosides, all othernucleosides are ribonucleosides and all internucleoside linkages for theAS strands are phosphorothioate and the internucleoside linkages for theS strand are phosphodiester.

SEQ ID NO: Sequence (5′-3′) 12 GUCAAAUCCAGAGGCUAGCAG 13CUGCUAGCCUCUGGAUUUGAC

The construct having overhangs was able to reduce PTEN mRNA levels toabout 36% of untreated control whereas the blunt ended construct wasable to reduce the PTEN mRNA levels to about 27% of untreated control.

Example 40

siRNA Hemimer Constructs

Three siRNA hemimer constructs were prepared and examined in a PTENassay. The hemimer constructs had 7, 2′-O-methyl nucleosides at the3′-end. The hemimer was put in the sense strand only, the antisensestrand only and in both strands to compare the effects.

SEQ ID NO:/ ISIS NO Constructs (overhangs) 14/271068 (S)5′-CAAAUCCAGAGGCUAGCAGUU-3′  9/ (AS) 3′-UUGUUUAGGUCUCCGAUCGUC-5′14/271068 (S) 5′-CAAAUCCAGAGGCUAGCAGUU-3′  9/ (AS)3′-UUGUUUAGGUCUCCGAUCGUC-5′ 14/ (S) 5′-CAAAUCCAGAGGCUAGCAGUU-3′  9/ (AS)3′-UUGUUUAGGUCUCCGAUCGUC-5′

Underlined nucleosides are 2′-O-methyl modified nucleosides, all othernucleosides are ribonucleosides and all internucleoside linkages for theAS strands are phosphorothioate and the internucleoside linkages for theS strand are phosphodiester.

SEQ ID NO: Sequence (5′-3′) 14 CAAAUCCAGAGGCUAGCAGUU

The construct having the 7, 2′-O-methyl nucleosides only in theantisense strand reduced PTEN mRNA levels to about 23% of untreatedcontrol. The construct having the 7, 2′-O-methyl nucleosides in bothstrands reduced the PTEN mRNA levels to about 25% of untreated control.When the 7, 2′-O-methyl nucleosides were only in the sense strand PTENmRNA levels were reduced to about 31% of untreated control.

Example 41

siRNA Vs asRNA Hemimers

Four hemimers were prepared and assayed as the asRNA's and also as thesiRNA's in a PTEN assay. The unmodified sequence was also tested as theasRNA and as the siRNA.

SEQ ID NO:/ ISIS NO Constructs (overhangs) 10/308746 (S)5′-AAGUAAGGACCAGAGACAAA-3′ 11/303912 (AS) 3′-UUCAUUCCUGGUCUCUGUUU-P 5′11/316449 (AS) 3′-UUCAUUCCUGGUCUCUGUUU-P 5′ 11/319013 (AS)3′-UUCAUUCCUGGUCUCUGUUU-P 5′ 11/319014 (AS) 3′-UUCAUUCCUGGUCUCUGUUU-P 5′11/319015 (AS) 3′-UUCAUUCCUGGUCUCUGUUU-P 5′

Underlined nucleosides are 2′-O-methyl modified nucleosides, all othernucleosides are ribonucleosides and all internucleoside linkages for theAS strands are phosphorothioate and the internucleoside linkages for theS strand are phosphodiester.

Construct siRNA (% mRNA) asRNA (% mRNA) 11/303912 21 32 11/316449 17 2611/319013 34 32 11/319014 54 42 11/319015 51 42

Percent mRNA is relative to untreated control in PTEN assay.

Example 42

Representative siRNA's Prepared Having 2′O-Me Gapmers

The following antisense strands of siRNA's were hybridized to thecomplementary full phosphodiester sense strand. Bolded monomers are2′-OCH₃ (also referred to as 2′-OMe) containing monomers. Underlinedmonomers have PS linkages. Monomers without underlines have PO linkages.

SEQ ID NO/ ISIS NO 15/300852 5′-OH-CUG CUA GCC UCU GGA UUU  (OMe/PO) GA15/300853 5′-P-CUG CUA GCC UCU GGA UUU GA (OMe/PO) 15/3008545′-OH-CUG CUA GCC UCU GGA UUU  (OMe/PO) GA 15/300855 5′-P-CUG  CUA   GCC UCU GGA UU U   GA (OMe/PO/ PS) 16/300856 5′OH-C UA   G CC UCU GGA UUU   GA (OMe/PO/ PS) 15/300858 5′-OH-CUG  CUA GCC UCU GGA UUU   (OMe/PS)GA 15/300859 5′-P-CUG  CUA GCC UCU GGA UUU   GA (OMe/PS) 16/3008605′-OH-C UA GCC UCU GGA UUU GA (OMe/PS) 17/303913 5′-OH-GUC UCU GGU CCU UAC UU (OMe/PS) 18/303915 5′-OH-U UU UGU CUC UGG UCC UU(OMe/PS) 19/303917 5′-OH-C UG GUC CUU ACU UCC CC (OMe/PS) 20/3087435′P-U UU GUC UCU GGU CCU UAC UU (OMe/PS) 21/308744 5′-P-UCU CUG GUC CUU ACU UCC CC (OMe/PS) 22/3287955′-P-UUU GUC UCU GGU CCU UAC UU (OMe/PS)

Example 43

Representative siRNA's Prepared Having 2′-O-Methyl Modified Nucleosides

The following antisense strands of siRNA's were hybridized to thecomplementary full phosphodiester sense strand. Where the antisensestrand has a TT 3′-terminus the corresponding sense strand also has a3′-TT (deoxyT's)

SEQ ID NO./ ISIS NO. 23/271065 CUG CUA GCC UCU GGA UUU GTT PO 24/271067CUG CUA GCC UCU GGA UUU GUU PO 25/271069 CUG CUA GCC UCU GGA UUU GUT PO23/271071 CUG CUA GCC UCU GGA UUU GTT PO 23/271072CUG CUA GCC UCU GGA UUU GTT PO 23/271073 CUG CUA GCC UCU GGA UUU GTT PO23/271074 CUG CUA GCC UCU GGA UUU GTT PO 23/271075CUG CUA GCC UCU GGA UUU GTT PO 23/271076 CUG CUA GCC UCU GGA UUU GTT PO23/271077 CUG CUA GCC UCU GGA UUU GTT PO 23/271078CUG CUA GCC UCU GGA UUU GTT PO 24/271079 CUG CUA GCC UCU GGA UUU GUU PO25/271081 CUG CUA GCC TCT GGA TTT GUU PO 26/271082CUG CUA GCC UCU GGA UUU GAC PO/PS 25/271083 CUG CUA GCC UCU GGA UUU GUUPO/PS 23/271084 CUG CUA GCC UCU GGA UUU GTT PO 23/283547CUG CUA GCC UCU GGA UUU GTT PO 23/293999 CUG CUA GCC UCU GGA UUU GTT PO23/294000 CUG CUA GCC UCU GGA UUU GTT PO 23/290223CUG CUA GCC UCU GGA UUU GTT PO

Example 44

Representative siRNA's Prepared Having 2′-F and 2′-OCH₃ Monomers

The following antisense strands of siRNA's were hybridized to thecomplementary full phosphodiester sense strand. Where the antisensestrand has a TT 3′-terminus the corresponding sense strand also has a3′-TT (deoxyT's). Bolded monomers are 2′-F containing monomers.Underlined monomers are 2′-OCH₃. Monomers that are not bolded orunderlined do not contain a sugar surrogate. Linkages are shown in theparenthesis after the sequence.

SEQ ID NO./ISIS NO. Composition (5′ 3′) Features 27/283546 CU G CU A GCC UCU GGA UUU GU.dT-3′ (OMe/F/PO) 28/336240 UUU GUC UCU GGU CCU UAC UU(OMe/F/PS)

1. A composition comprising a first oligomer and a second oligomer,wherein: at least a portion of said first oligomer is hybridized with atleast a portion of said second oligomer, at least a portion of saidfirst oligomer is complementary to a selected target nucleic acid, eachof said first and second oligomers are chimeric oligomers, wherein thesecond oligomer is a blockmer comprising at least one XXYYXX motif, andwherein each X is an unmodified ribonucleoside and each Y is anucleoside having a 2′-OCH₃ substituent group.
 2. The composition ofclaim 1 wherein each of said first and second oligomers comprise fromabout 18 to about 30 linked nucleosides.
 3. The composition of claim 1wherein each of said first and second oligomers comprise from about 21to about 24 linked nucleosides.
 4. The composition of claim 1 whereineach of said first and second oligomers comprise 21 linked nucleosides.5. The composition of claim 1 wherein each nucleoside of said first andsecond oligomers is independently selected from an unmodifiedribonucleoside, a nucleoside having a 2′-OCH₃ substituent group, and a2′-deoxythymidine nucleoside.
 6. The composition of claim 1 wherein saidfirst oligomer is an antisense oligomer and said second oligomer is asense oligomer.
 7. The composition of claim 1 wherein said first andsaid second oligomers are a complementary pair of siRNA oligomers. 8.The composition of claim 2 wherein each internucleoside linkage linkingthe linked nucleosides is, independently, a phosphodiester orphosphorothioate internucleoside linkage.
 9. The composition of claim 2wherein each internucleoside linkage linking the linked nucleosides is aphosphodiester internucleoside linkage.
 10. The composition of claim 1wherein the 5′-terminal nucleoside of said first oligomer has a5′-phosphate group.
 11. The composition of claim 1 wherein at least oneof said first or second oligomers has an overhang.
 12. The compositionof claim 1 wherein each of said first and second oligomers comprises a3′-overhang consisting of two 2′-deoxythymidine nucleosides.
 13. Thecomposition of claim 1 comprising at least two of said XXYYXX motifs.14. The composition of claim 1, wherein said first oligomer does notcomprise an internal DNA segment.
 15. The composition of claim 1,wherein said first oligomer is not a deoxy gapmer.